(Received for publication, June 28, 1995)
From the
The platelet-derived growth factor (PDGF) A-chain has been implicated in the initiation and progression of vascular occlusive lesions. The elements in the human PDGF-A promoter that mediate increased expression of the gene in vascular endothelial cells have not been identified. A potent inducer of PDGF-A expression in endothelial cells is phorbol 12-myristate 13-acetate (PMA). 5`-Deletion and transfection analysis revealed that a G+C-rich region in the proximal PDGF-A promoter is required for PMA-inducible gene expression. This region bears overlapping consensus recognition sequences for Sp1 and Egr-1. PMA induces Egr-1 mRNA expression within 1 h, whereas PDGF-A transcript levels increase after 2-4 h. Constitutive levels of Sp1 are not altered over 24 h. A specific nucleoprotein complex is formed when an oligonucleotide bearing the G+C-rich element is incubated with nuclear extracts from PMA-treated cells. The temporal appearance of this complex is consistent with the transient increase in Egr-1 transcripts. Antibodies to Egr-1 completely supershift the PMA-induced complex. Interestingly, increased nuclear levels of Egr-1 attenuate the ability of Sp1 to interact with the oligonucleotide, implicating competition between Egr-1 and Sp1 for the G+C-rich element. Binding studies with recombinant proteins demonstrate that Egr-1 can displace Sp1 from this region. Insertion of the G+C-rich element into a hybrid promoter-reporter construct confers PMA inducibility on the construct. Mutations that abolish Egr-1 binding also abrogate expression induced by PMA or overexpressed Egr-1. These findings demonstrate that PMA-induced Egr-1 displaces Sp1 from the G+C-rich element and activates expression driven by the PDGF-A proximal promoter in endothelial cells. The Sp1/Egr-1 displacement mechanism may be an important regulatory circuit in the control of inducible gene expression in vascular endothelial cells.
The platelet-derived growth factor (PDGF) ()is a
potent mitogen that has been implicated to play a role in a diversity
of normal and pathological settings (for reviews see Heldin(1992),
Khachigian and Chesterman(1993), and Ross(1993)). PDGF occurs as a
homodimer or heterodimer of A- and B-chains covalently linked by
disulfide bonds and has a relative molecular mass of approximately 28
kDa. The biological response to PDGF is mediated by high affinity
cell-surface receptors with split tyrosine kinase domains. The
-receptor is bound by both polypeptide chains of PDGF, whereas the
-subunit is bound with high affinity only by the B-chain (Heldin et al., 1988). PDGF A-chain mRNA has been detected in a
variety of cells of human origin in culture (Betsholtz et al.,
1986; Raines et al., 1990) and undergoes alternative splicing
of exons 2 (Sanchez et al., 1991) and 6 (Collins et
al., 1987; Tong et al., 1987). Three transcript species
can be generated from the human PDGF-A gene (Bonthron et al.,
1988), which are derived from the same transcriptional start site
(Takimoto et al., 1991) and arise from the utilization of
alternative poly(A) sites in exon 7 (Bonthron et al., 1988).
Somatic cell hybrid chromosome segregation analysis (Betsholtz et
al., 1986) and in situ hybridization using endothelial
cell-derived cDNA (Bonthron et al., 1988) or non-isotopic
techniques and genomic clones (Bonthron et al., 1992) assigned
the gene to chromosome 7 (7p21-p22).
Isolation of genomic clones encoding the human PDGF A-chain gene allowed characterization of its structural organization and facilitated subsequent investigation of the molecular mechanisms controlling transcription of the gene (Bonthron et al., 1988; Rorsman et al., 1988; Takimoto et al., 1991). The human PDGF A-chain gene spans approximately 24 kilobases of genomic DNA and has a single transcriptional start site located 36 bp downstream of the TATA box (Bonthron et al., 1988). The minimal promoter region, sufficient for optimal promoter activity, consists of approximately 150 bp in human epithelial carcinoma (HeLa) cells (Lin et al., 1992) and 120 bp in African green monkey renal epithelial (BSC-1) cells (Kaetzel et al., 1994). This region, rich in G+C content, is hypersensitive to cleavage by S1 nuclease (Wang et al., 1992a) and contains potential overlapping recognition elements for the zinc-finger transcription factor, Sp1 (Dynan and Tjian, 1983; Kadonaga et al., 1987), the immediate-early growth response gene product, Egr-1 (Krox-24, zif268) (Rauscher et al., 1990), and the Wilms' tumor suppressor gene product, WT-1 (Rauscher et al., 1990). Although this G+C-rich sequence in the PDGF-A promoter is bound by nuclear proteins from cultured BSC-1 cells (Kaetzel et al., 1994), and Egr-1 and WT-1 overexpression can modulate promoter activity (Gashler et al., 1992; Wang et al., 1992b), it is unclear whether Sp1, Egr-1, or WT-1 actually play a physiologic role in the transcriptional regulation of PDGF-A. Several upstream promoter regions have also been implicated in the induced expression of the gene. First, a novel element located between bp -102 and -82 interacts with nuclear protein(s) (PDGF-A-BP-1) in human mesangial cells exposed to phorbol 12-myristate 13-acetate (PMA) (Bhandari et al., 1995). Second, a consensus serum response element between bp -477 and -468 mediates PDGF-induced reporter gene expression in human foreskin fibroblasts (Lin et al., 1992). The PDGF-A promoter is also subject to negative transcriptional regulation. Overexpression of WT-1 represses PDGF-A promoter-dependent reporter gene expression in murine fibroblasts and human embryonic kidney cells (Gashler et al., 1992; Wang et al., 1992b). Additionally, 5`-deletion analysis in BSC-1 cells defined two negative regulatory regions located between bp -1800 and -1029 and bp -1029 and -880 (Kaetzel et al., 1994). The identity of the endogenous nuclear transcription factors that actually interact with any of the regulatory regions in the PDGF-A promoter has not been determined.
In this report, PMA was used as a model agonist to define specific nuclear transcription factors that interact with functional nucleotide elements in the proximal PDGF-A promoter in cultured vascular endothelial cells. Using 5`-deletion and transient transfection analysis, we demonstrate the requirement for the region between bp -71 and -55 for PMA-inducible expression. This region bears overlapping Egr-1 and Sp1 binding elements. Northern blot analysis reveals that PMA induces Egr-1 expression with a time course earlier than PDGF-A, whereas Sp1 transcript levels are unchanged. Gel shift and supershift studies demonstrate that both PMA-induced and recombinant Egr-1 bind to the G+C-rich element by displacing Sp1. Mutations that abolish the ability of Egr-1 to interact with the proximal PDGF-A promoter also abrogate expression induced either by PMA or overexpressed Egr-1. Thus, PMA-induced expression of the PDGF A-chain in endothelial cells is mediated, at least in part, by the rapid and transient induction of Egr-1 and involves an interchange of factors occupying the G+C-rich element in the proximal promoter. This displacement mechanism may be a common theme in the induced expression of the PDGF A-chain gene by multiple signals in a variety of biological settings.
Binding reactions using
[P]-A were carried out in a total volume of 20
µl containing 2 µg of poly(dI
dC)-poly(dI
dC)
(Sigma), 0.5% sucrose in 10 mM Tris-HCl, pH 8, 50 mM
MgCl
, 1 mM EDTA, 1 mM DTT, 5% glycerol,
20 µg of bovine serum albumin, and 1 mM PMSF and
increasing amounts of recombinant protein. Following an incubation for
1 h at 4 °C, 20 µl of DNase buffer (25 mM NaCl, 10
mM HEPES, pH 7.9, 5 mM MgCl
, 1
mM CaCl
) containing 0.02 units of DNase I
(Promega) was added and allowed to digest for 5 min at this
temperature. 10 µl of stop solution (150 mM EDTA, 5% SDS,
250 µg/ml salmon sperm DNA) was added to each tube, and
phenol/chloroform was extracted prior to DNA precipitation with
absolute ethanol. The pellets were washed with 70% ethanol, evaporated
to dryness, and resuspended in sequencing sample buffer. The samples
were boiled for 2 min and applied to an 8% sequencing gel and run at
1500 V. The gels were dried and autoradiographed overnight at -80
°C.
Figure 1:
Northern blot analysis of vascular
endothelial cells exposed to PMA. RNA was isolated either from
unstimulated BAEC or cells that were incubated with 100 ng/ml PMA in
normal growth medium for 0, 1, 2, 4, 6, 9, 12, 16, 20, or 24 h at 37
°C. Samples were normalized for total RNA (15 µg) prior to
electrophoresis and transfer to a nylon membrane. The blots were
hybridized with specific P-labeled cDNA probes, and
binding was assessed by autoradiography as described under
``Experimental Procedures.''
Figure 2: Effect of PMA on reporter gene expression driven by 5`-deletions of the human PDGF A-promoter. BAEC were transiently transfected with 15 µg of each construct and 2 µg of normalizing plasmid (pTKGH) using the calcium phosphate precipitation technique. The cells were incubated with 100 ng/ml PMA for 24 h at 37 °C. CAT activity (arbitrary units) was normalized to levels of human growth hormone in the supernatant as described under ``Experimental Procedures.''
PMA-induced CAT
activity was detected in cells that were transfected with constructs
pACCATXho, e33, and f28 (Fig. 2). Construct f36, however,
was unable to mediate PMA-inducible gene expression (Fig. 2).
The sequence between the 5`-deletion end points of f28 and f36
corresponds to a G+C-rich element that contains overlapping
consensus nucleotide recognition sequences for the zinc-finger
transcription factors Sp1 and Egr-1 (Fig. 3A).
Cotransfection studies indicate that constructs e33 and f28 respond to
overexpressed Sp1 and Egr-1, whereas f36 does not. (
)Thus,
basal and PMA-induced expression mediated by the PDGF-A promoter in
endothelial cells requires the proximal G+C-rich element and may
involve the participation of Sp1 and/or Egr-1. The relative increase in
reporter activity in response to PMA is consistent with -fold increases
in CAT activity reported elsewhere (Angel et al., 1988; Fazio et al., 1991; John et al., 1995).
Figure 3:
Interaction of the G+C-rich element
in the proximal PDGF-A promoter with nuclear proteins from endothelial
cells exposed to PMA. A, oligonucleotide A bearing the
nucleotide sequence between bp -76 and -47 in the human
PDGF-A promoter. The putative Sp1 and Egr-1 binding sites are
indicated. B, specific interaction of endothelial nuclear
proteins with the A-chain promoter. EMSA was performed using P-oligonucleotide A and nuclear extracts from unstimulated
BAEC or cells exposed to 100 ng/ml PMA for 1 h at 37 °C. C, time course appearance of complex A3. EMSA was performed
using
P-oligonucleotide A and nuclear extracts from cells
exposed to 100 ng/ml PMA for 0, 1, 2, 4, 9, 12, and 24 h at 37 °C. D, supershift analysis using nuclear extracts of cells exposed
to 100 ng/ml PMA for 1 h. The binding reaction contained 1 µl of
the antibody indicated. Complexes A1-A5 are indicated by arrows. Binding and electrophoresis were carried out as
described under ``Experimental
Procedures.''
Polyclonal antipeptide antibodies were used to determine the identity of the nuclear protein(s) involved in the PMA-induced complex. A complete supershift of complex A3 was obtained using antibodies directed toward Egr-1 (Fig. 3D). Thus, Egr-1 interacts with the PDGF-A G+C-rich element in cultured endothelial cells within 1 h of exposure to PMA. AP-2 and WT-1 are zinc-finger transcription factors that interact with the G+C-rich nucleotide sequences, 5`-(C/T)(C/G)(C/G)CC(C/A)N(G/C)(G/C)(G/C)-3` (Imagawa et al., 1987; Williams et al., 1989) and 5`-GCGTGGGAGT-3` (Nakagama et al., 1995), respectively. Antibodies to AP-2, WT-1, or to PEA-3, however, failed to produce a supershift (Fig. 3D).
Figure 4:
Induction of Egr-1 by PMA attenuates the
ability of Sp1 to interact with the A-chain promoter oligonucleotide.
EMSA was performed using P-oligonucleotide A and nuclear
extracts from BAEC exposed to 100 ng/ml PMA for 1 h at 37 °C.
Complexes A1-A5 are indicated by the arrows. S denotes a supershift and is indicated by the arrow.
Nuclear extracts were corrected for protein concentration prior to
assay. Binding and electrophoresis were carried out as described under
``Experimental Procedures.''
Figure 5:
Interaction of recombinant Sp1 and Egr-1
with the proximal A-chain promoter. EMSA using Sp1 (A) or
Egr-1 (B) in a binding reaction with P-oligonucleotide A. C, in vitro DNase I
footprinting using Egr-1 with the single end-labeled PDGF-A probe
([
P]-A). [
P]-A was
incubated with increasing amounts of recombinant Egr-1 for 60 min at 4
°C. DNase I (0.02 units) was added to the binding reaction and
incubated for 5 min at this temperature. Electrophoresis was carried
out as described under ``Experimental Procedures.'' Temporal
stability of nucleoprotein complexes involving Sp1 (D) and
Egr-1 (E). Bound complexes and free probe are indicated by arrows. Recombinant proteins were incubated with
P-oligonucleotide A for 30 min at 22 °C and incubated
with a 1000-fold molar excess of unlabeled oligonucleotide A for the
periods indicated. The binding mixture was applied to a running
non-denaturing polyacrylamide gel at the times indicated.
Electrophoresis was carried out as described under ``Experimental
Procedures.''
To provide an
indication of the stability of the nucleoprotein complexes involving
Sp1 and Egr-1 over time, EMSA was carried out using the running gel
technique (Franzoso et al., 1993). Recombinant Sp1 was
incubated with P-oligonucleotide A for 30 min at 22 °C
and applied to a non-denaturing polyacrylamide gel following various
times. The Sp1-
P-oligonucleotide A complex was apparent
even after 150 min at this temperature (Fig. 5D).
Addition of 1000-fold molar excess of unlabeled oligonucleotide A
displaced Sp1 from
P-oligonucleotide A within 30 min (Fig. 5D). When recombinant Egr-1 was used instead of
Sp1, the nucleoprotein complex was similarly apparent after 150 min (Fig. 5E). Unlike Sp1, however, addition of excess
unlabeled oligonucleotide A resulted only in partial displacement of
the prebound Egr-1 (Fig. 5E). These results indicate
that both Sp1 and Egr-1 bind to the PDGF-A oligonucleotide in a stable
manner. The apparent off-rate of Egr-1, however, is slower than that of
Sp1.
Figure 6:
Interplay of recombinant Sp1 and Egr-1 at
the proximal PDGF-A promoter. A, displacement of prebound Sp1
by Egr-1 using P-oligonucleotide A and running EMSA.
Recombinant Sp1 was incubated with
P-oligonucleotide A for
30 min at 22 °C. A 40-fold molar excess of Egr-1 was added, and the
mixture was applied to a non-denaturing polyacrylamide gel at the times
indicated. Bound complexes and free probe are indicated by arrows.
B, displacement of prebound Sp1 with Egr-1 using the
P-labeled footprint probe ([
P]-A)
in EMSA. [
P]-A was incubated with a fixed amount
of Sp1 or buffer alone for 10 min. Increasing amounts of recombinant
Egr-1 were added and incubated for a further 20 min. Binding and
electrophoresis were carried out as described under ``Experimental
Procedures.''
Figure 7:
Mutation of the Egr-1 binding site in the
proximal PDGF-A promoter abolishes expression inducible by PMA or
Egr-1. A, EMSA was performed using recombinant Egr-1 with
either P-oligonucleotide A or its mutant
([
P]-Oligo Am). BAEC were transiently
transfected with 10 µg of SV40-based heterologous promoter CAT
reporter constructs bearing either wild-type (A.SV40-CAT) or mutant
(Am.SV40-CAT) PDGF-A promoter sequences. The cells were either
incubated with 100 ng/ml PMA for 24 h at 37 °C (B) or
cotransfected with the Egr-1 expression vector, CMV.Egr-1 (C).
CAT activity (arbitrary units) was normalized to levels of human growth
hormone in the supernatant as described under ``Experimental
Procedures.''
The present report indicates that the induction of PDGF
A-chain expression in endothelial cells by PMA involves the interplay
of zinc-finger transcription factors with elements in the core
promoter. A schematic representation of these events appears in Fig. 8. Several lines of evidence support this model. First,
5`-deletion analysis of the PDGF-A promoter indicates that Sp1 elements
are required for basal expression in endothelial cells (Fig. 2),
consistent with findings in other cell types (Kaetzel et al.,
1994; Lin et al., 1992). The actual number of Sp1 sites
required for basal activity is not clear. However, single point
mutations in individual putative Sp1 sites did not reduce PDGF-A
promoter activity, ()suggesting that not all Sp1 sites are
required for basal expression. Second, recombinant Sp1 interacts with
the G+C-rich element in a specific (Fig. 5A),
dose-dependent (Fig. 5A) (Kim et al., 1989),
and stable manner (Fig. 5D). Third, EMSA, using nuclear
extracts from unstimulated cells, indicates that the G+C-rich
element in the proximal PDGF-A promoter is occupied by Sp1 (Fig. 4). Finally, the constitutive expression of Sp1 transcript (Fig. 1) and protein
is not affected by PMA. Sp1,
which was first identified in HeLa cells by virtue of its ability to
activate the SV40 early promoter (Dynan and Tjian, 1983), mediates the
basal expression of the structurally similar PDGF-B gene in cultured
endothelial cells (Khachigian et al., 1994).
Figure 8: Model for the displacement of Sp1 by Egr-1 in endothelial cells exposed to PMA. Sp1 binds to the proximal PDGF-A promoter and mediates basal expression of the gene. Egr-1 induced by PMA displaces Sp1 from the G+C-rich element and stimulates PDGF-A gene expression. Although the actual molecular stoichiometry of Sp1 and Egr-1 occupying the proximal promoter is unclear, three Sp1 and two Egr-1 molecules are represented in the model based on the number of consensus elements in this region (Fig. 3A) and higher order binding (Fig. 5, A and B). The nuclear protein A5, which interacts with an undefined site in the proximal promoter, is not displaced by PMA-induced Egr-1 (Fig. 3B).
Upon exposure of endothelial cells to PMA, Egr-1 is induced and binds to the G+C-rich element in the proximal promoter by displacing Sp1. Several findings support this part of the model (Fig. 8). First, EMSA and in vitro DNase I footprinting studies indicate that like Sp1, recombinant Egr-1 interacts with the G+C-rich element in a specific (Fig. 5B), dose-dependent (Fig. 5, B and C), and stable (Fig. 5E) manner. The number of functional Egr-1 sites required for induced PDGF-A expression is not clear. Second, Egr-1 mRNA is dramatically induced in endothelial cells within minutes of exposure to PMA (Fig. 1). Third, the time course of induction of Egr-1 mRNA is virtually identical to appearance of the nucleoprotein complex A3 (Fig. 3C). Fourth, supershift analysis indicates that PMA-induced Egr-1 attenuates the ability of Sp1 to interact with the promoter (Fig. 4). Finally, a molar excess of Egr-1 can displace prebound Sp1 from this region (Fig. 6, A and B). It is unlikely that Egr-1 and Sp1 interact simultaneously with the G+C-rich element in the proximal PDGF-A promoter. The presence of both Egr-1 and Sp1 in the same EMSA reaction does not result in a super complex (Fig. 6, A and B), and antibodies to Egr-1 or Sp1 do not supershift a single band from endothelial cell nuclear extracts (Fig. 4). Collectively, these findings indicate that PMA-induced Egr-1 stimulates gene expression driven by the PDGF-A promoter in endothelial cells by displacement of Sp1 from the G+C-rich element.
The molecular mechanism(s)
responsible for the return of PDGF-A transcript mRNA to basal levels
following induction by PMA are unclear. It is tempting to speculate,
however, that one or more of the following transcriptional events may
be involved. First, the transient increase in Egr-1 transcript (Fig. 1), protein, and DNA binding activity (Fig. 3B) suggests that Sp1 may reoccupy the
G+C-rich element when the stoichiometric ratio of Sp1 to Egr-1
returns to preinduction levels, greatly favoring Sp1. Second, Egr-1
could be actively displaced by a third transcription factor such as
WT-1. Recombinant WT-1 has been shown previously to interact with the
same G+C-rich element in the proximal PDGF-A promoter (Gashler et al., 1992; Wang et al., 1992b). We did not detect
a nucleoprotein complex involving endothelial WT-1 by supershift
analysis; however, a WT-1-like transcription factor may be involved in
the post-induction transcriptional repression of PDGF-A. Finally, other
regulatory proteins could displace Egr-1 from the G+C-rich element
via direct protein-protein interactions. For example, the activation of
creatine kinase gene expression by WT-1 is repressed by physical
association with p53 (Maheswaran et al., 1993). Interestingly,
an active transcriptional repression mechanism has also been suggested
in the down-regulation of PDGF-A gene expression after serum
stimulation (Takimoto and Kuramoto, 1995).
The regulatory events
presented in this manuscript indicate that the complex process of
transcriptional activation can involve the interchange of transcription
factors associating with distinct promoter elements (Tjian and
Maniatis, 1994). Interestingly, overlapping putative nucleotide
recognition elements for Egr-1 and Sp1 appear in the promoters of human
tissue factor (Mackman et al., 1989), transforming growth
factor-1 (Kim et al., 1989; Dey et al., 1994),
colony-stimulating factor-1 (Harrington et al., 1993), tumor
necrosis factor (Kramer et al., 1994), murine thrombospondin-1
(Shingu and Bornstein, 1994), acetylcholinesterase (Mutero et
al., 1995), and in the Egr-1 promoter itself (Cao et al.,
1993). Although binding and cotransfection studies have implied a
functional role for Sp1 and Egr-1 in the regulation of certain of these
genes, actual competition between these factors has not been
demonstrated. Accordingly, the exchange of Sp1 and Egr-1 at overlapping
motifs may be an important common event in the control of inducible
gene expression.