(Received for publication, September 8, 1995; and in revised form, December 5, 1995)
From the
Thrombin is a coagulation system protease that also serves as a potent stimulator of gene expression in several cell types, including endothelial cells (EC). We and others have previously demonstrated that the transcription of platelet-derived growth factor (PDGF) B-chain (c-sis) by EC is stimulated severalfold by thrombin. Here we examine the molecular mechanism of this regulatory process using bovine aortic EC transiently transfected with a vector containing the chloramphenicol acetyltransferase (CAT) gene under the control of a 400-base pair fragment of the human PDGF B-chain promoter. Thrombin treatment of these cells caused a severalfold increase in CAT expression. Deletion analysis and site-directed mutagenesis revealed that the region spanning nucleotides -61 to -53 from the transcription initiation site (referred to as the thrombin response, or ThR, region) was critical for the transcriptional response to thrombin. Electrophoretic mobility shift assays with an oligonucleotide corresponding to the region -64 to -44, which contained the ThR region, led to the identification of a thrombin-inducible nuclear factor (TINF) in extracts from thrombin-treated, but not control, EC. TINF was formed as early as 40 min post-thrombin treatment, persisted for at least 7 h, but was no longer present after 24 h. TINF appeared in the absence of de novo protein synthesis. The ThR region consists of a repeat of a CCACCC element in an ABBA configuration, which, based on mutation analysis and transfection assays, appears to be critical in mediating thrombin stimulation of the PDGF B-chain gene. The conservation of the ThR region in the promoter of the PDGF B-chain among three species (human, feline, and murine) further supports the importance of this region as a cis-acting regulatory element.
Endothelial cell (EC) ()injury or activation and the
subsequent proliferation of vascular smooth muscle cells (SMC) are
central to the development of atherosclerosis(1) . While the
triggering molecule(s) responsible for stimulating the multiplication
of SMC is not known, a viable candidate is platelet-derived growth
factor (PDGF), the major mitogen in human serum (2, 3, 4, 5) . PDGF, although
originally purified from human platelets, is produced in a regulated
manner by numerous transformed cells and by several types of normal
diploid cells, including EC, SMC, mesangial cells, activated
macrophages, and cytotrophoblasts(6, 7, 8) .
PDGF consists of a disulfide-linked heterodimer or homodimer of two
distinct but homologous subunits designated the A- and
B-chains(6) . The B-chain is the protein product of the
c-sis proto-oncogene(9, 10) .
Regulated EC
expression of PDGF production in vivo may be important in
stimulating proliferation of the underlying perivascular cell types and
in the recruitment of both leukocytes and SMC in response to vascular
injury(11, 12) . A probable physiological modulator of
PDGF release from the endothelium is -thrombin, a multifunctional
serine protease generated at sites of vascular injury, which has been
demonstrated in vitro to induce the expression and release of
PDGF A- and B-chains from
EC(13, 14, 15, 16, 17) .
Thrombin, at physiologically relevant concentrations, stimulates a
number of EC functions including the generation of
prostacyclin(18) , platelet-activating factor(19) , von
Willebrand factor(20) , plasminogen activator(21) , and
its inhibitor(22) . The molecular mechanism underlying thrombin
stimulation of transcription of the PDGF B-chain gene or, in fact,
thrombin induction of any gene in EC, as well as in any other cell
type, remains unknown.
Regulation of PDGF B-chain gene transcription has been explored in multiple cell types. Specific sequences have been defined within the 5` untranslated region of the B-chain gene, which may be important in the regulation of its transcription(23) . Transfection experiments in K-562 cells (a hematopoietic cell type) have defined a minimal promoter region which includes the sequence extending 400 bp 5` of the transcription initiation site(24) . In addition, several DNase I-hypersensitive sites have been located in the human PDGF B-chain gene, although localization of these sites has focused on the first intron and the region downstream of the coding sequence with little known about the 5` flanking region(25, 26) . Recently, Kachigian et al.(27) have defined a minimal promoter for basal expression of the PDGF B-chain gene in EC. This promoter region contains several consensus sequences for binding such transcriptional factors as Ets family members and AP-1 complexes. These investigators, however, have not examined the role of any of these elements in agonist-stimulated transcription of this gene in EC. Others have used linker scanning of the PDGF B-chain core promoter to identify an element essential for TPA-induced activation of this gene in K562 cells (28) .
In this report, we present evidence supporting the identification of a 9-bp region in the PDGF B-chain promoter as being responsible for thrombin-induced transcription of this gene. We further demonstrate that a thrombin-inducible nuclear factor (TINF) and the transcription factor Sp1 are two distinct factors that bind to this region of DNA.
Figure 1: PDGF B-chain promoter region and its response to thrombin. Bovine aortic EC were transfected with the p400 PDGF B-chain promoter-CAT construct as described under ``Experimental Procedures.'' Cells were then treated with the indicated concentrations of thrombin for 18 h. Cell extracts were prepared and assayed for CAT activity as described. Values represent the mean of duplicate cultures. Results are representative of those obtained with three strains of EC and two lots of thrombin.
Figure 2: Identification of the ThR in the PDGF B-chain promoter using deletion analysis. A, schematic diagram indicating the location of the deletion constructs in the PDGF B-chain promoter. Each construct label indicates the remnant size (bp) of the promoter region cloned into the expression vector (based on the transcription initiation site described by Pech et al. in (24) ). TIS indicates the transcription initiation site. B, bovine aortic EC were transfected with the deletion mutant constructs depicted in A, treated with or without thrombin (10 units/ml) for 16 h, and CAT activity was determined. Results represent mean of triplicate cultures ± S.E. Figure is representative of more than four independent experiments.
To further identify the specific sequence within the p86 construct of the PDGF B promoter responsible for thrombin induction, we employed an alternative approach. Transcription factors present in the nuclear extracts of thrombin-treated, but not in untreated EC, should bind to their specific DNA sequence, allowing visualization by EMSA. Oligonucleotides corresponding to two segments of the region containing the thrombin response element, one corresponding to the 5` half, designated A (nucleotides -79 to -66) and the second, the 3` half, designated B (nucleotides -64 to -44) were synthesized. An oligonucleotide corresponding to the -43 to -31 sequence, outside of the thrombin response region, designated C was prepared as a control (Fig. 3). Each oligomer was radiolabeled for use in EMSA. Incubation of oligonucleotide A or C with nuclear extracts prepared from bovine aortic EC that had been treated with thrombin did not reveal any novel shifted bands compared with nuclear extracts from untreated EC (data not shown). The B oligomer, however, yielded a thrombin-dependent DNA-protein complex that migrated more rapidly than several other complexes that were constitutively present in nuclear extracts (Fig. 4). The specificity of these DNA-protein complexes was demonstrated by competition experiments with 100-fold molar excess of unlabeled oligonucleotides, representing several consensus binding site sequences for known transcription factors as well as other sequences that are proximal to the B region (Fig. 4). These oligonucleotides are fully defined in Table 1. The competition study showed that among the many oligomers tested, only an excess of the unlabeled oligonucleotide B competed efficiently with labeled B oligomer in binding to TINF.
Figure 3: Region of the PDGF B-chain promoter containing the thrombin response element. Diagram showing the sequence of the region involved in thrombin responsiveness. Oligonucleotide sequences used as probes for EMSA are shown in boxes. The arrows indicate the beginning position of the PCR constructs. The ThR element is underlined.
Figure 4:
Characterization of the thrombin response
region by EMSA. Nuclear extracts prepared from bovine aortic EC treated
without (control) or with thrombin (10 units/ml) for times indicated
were used in EMSA. The 5` end-labeled B sequence oligomer (see Fig. 3) was incubated with nuclear extracts (5 µg) in the
absence or presence of a 100-fold molar excess of unlabeled
oligonucleotides corresponding to the B region, C region, A region, AP1
binding sequence, NFB binding sequence, AP2 binding sequence,
TFIID binding sequence, and the CREB binding sequence. The TINF complex
is indicated.
To demonstrate that the B region was a functional thrombin response element, we generated a construct that contained the B region attached to the thrombin-unresponsive minimal promoter region p43 (Fig. 2B) and assayed for thrombin-stimulated CAT activity in EC. This new PCR-generated construct, designated p64, was linked to the CAT reporter gene and used for transfection into bovine aortic EC. As shown in Fig. 5, EC containing the p64 construct responded to thrombin with greater than a 3-fold increase in CAT expression over similarly transfected controls that were not treated with thrombin, and this level was comparable to cells containing the fully thrombin-responsive p86 construct. In addition, thrombin treatment of the p64 construct-transfected EC yielded 3 times the -fold induction in CAT activity than was seen with the p43 construct-transfected EC, indicating that the B region was sufficient to restore thrombin responsiveness to the p43 construct. Taken together, these data strongly suggested that the B region contained the element(s) necessary to increase the transcription of the PDGF B-chain gene as a result of thrombin stimulation.
Figure 5: Functional analysis of the thrombin response region. Bovine aortic EC were transfected with the p43 and p64 CAT constructs as well as p86-CAT as positive control, treated with or without thrombin (10 units/ml) for 16 h, and CAT activity was determined as described under ``Experimental Procedures.'' Results represent mean of triplicate cultures ± S.E. Figure is representative of four independent experiments. -Fold induction of CAT activity is expressed as a ratio of cpm of thrombin-treated cultures/cpm of media-treated cultures.
Figure 6: Time course of TINF appearance in EC nuclear extracts. Nuclear extracts prepared from bovine aortic EC treated without (control) or with thrombin (10 units/ml) for the times indicated were heat treated (10 min, 47 °C) to eliminate Sp1 binding and then incubated with labeled double-stranded oligomer B and used in EMSA. Arrow indicates the position of the TINF complex.
In an attempt to identify other nuclear proteins in thrombin-stimulated EC that bind to the B region and to determine the novelty of TINF, competition assays were performed using oligonucleotides containing consensus binding site sequences for several known nuclear factors. As shown in Fig. 4, many consensus sequence oligonucleotides failed to cause any reduction in the TINF-DNA complex. Effective competition was observed, however, with a 100-fold molar excess of unlabeled oligomer representing the consensus binding site for Sp1 (Fig. 8A). This result suggested that in addition to the TINF complex, Sp1-like proteins may bind to the B region. To determine whether TINF and Sp1 are distinct proteins, a ``supershift'' experiment was performed in which Sp1 antibody, Egr-1 antibody, or purified IgG was incubated with the nuclear extract-labeled oligomer mixture prior to the mobility shift assay. One of the major constitutively present bands (band 1) was shifted to a lower mobility in the gel (band ss-Sp1) with the Sp1 antibody, whereas TINF migration was unaltered (Fig. 7). The Egr-1 antibody as well as the IgG control had no effect on any of the bands from the EC extracts. Evidence that TINF is distinct from Sp1 includes its gel mobility, which is greater than the Sp1 proteins, its insensitivity to heat treatment (47 °C for 10 min; data not shown)(35) , and the inability of an Sp1 polyclonal antibody to either deplete nuclear extracts of TINF or to supershift TINF in the EMSA.
Figure 8: Characterization of the specificity of TINF complex formation by competition in EMSA. Nuclear extracts were prepared from bovine aortic EC treated without (control) or with thrombin (10 units/ml) for 5 h. The 5` end-labeled B oligonucleotide was incubated with nuclear extracts (5 µg) in the absence or presence of 100-fold molar excess of various mutated B oligonucleotides as indicated. EMSA was performed as described under ``Experimental Procedures.'' Arrow indicates the position of the TINF complex.
Figure 7: Supershift evidence that TINF and Sp1 are distinct proteins. Nuclear extracts were prepared from bovine aortic EC treated without (control) or with thrombin (10 units/ml) for 5 h. Labeled double-stranded oligomer B was incubated with nuclear extracts (5 µg) from EC as described under ``Experimental Procedures'' and then incubated with 1 µl of medium, anti-Sp1 antiserum, anti-Egr-1 antiserum, or IgG prior to electrophoresis. SS refers to the supershifted Sp1 band.
Figure 9: Characterization of the specificity of TINF complex formation by competition in EMSA by use of radiolabeled mutated oligomers. Nuclear extracts were prepared from bovine aortic EC treated without (control) or with thrombin (10 units/ml) for 5 h. Labeled double-stranded wild type oligomer (B) or mutated oligomers (B mutant and S-610587), were incubated with EC nuclear extracts (5 µg). EMSA was performed as described under ``Experimental Procedures.'' Arrow indicates the position of the TINF complex.
To confirm that the CCACCC element is mediating thrombin-induced transcription of the PDGF B-chain gene in bovine aortic EC, we generated several mutants of the PDGF B-chain promoter by PCR and ligated them into the CAT expression vector in order to perform functional analyses. These mutants included a p64 mutant plasmid, in which the CCACCCACC (ThR region) sequence was fully substituted by AAGTTTGAA, and p64S610587, in which four base substitutions were made within the CCACCC element. These mutant plasmid constructs were used to transfect bovine aortic EC, and CAT activity was measured in replicate cultures following treatment with thrombin or no stimulator. Cells transfected with the mutant constructs did not demonstrate thrombin responsiveness, although EC transfected with either of two wild-type constructs, p86 and p64, exhibited a severalfold increase in CAT activity in response to thrombin (Fig. 10). These results further support a role for the CCACCC element in thrombin stimulation of PDGF B-chain gene expression.
Figure 10: Transfection assay using PDGF B-chain promoter-CAT constructs with mutations in the thrombin response region. Bovine aortic EC were transfected with the p86, p64, p64 mutant, and p64S610587 PDGF B-chain promoter constructs, treated with or without thrombin (10 units/ml) for 16 h, and CAT activity was determined as described under ``Experimental Procedures.'' Results represent mean of triplicate cultures ± S.E. Figure is representative of at least six independent experiments. -Fold induction of CAT activity is expressed as a ratio of cpm of thrombin-treated cultures/cpm of media-treated cultures.
We have employed a combination of reporter gene construct-transfection experiments and gel shift assays to map the region of the PDGF B-chain gene that controls the transcriptional response to thrombin in EC. The ThR region is localized to nucleotides -61 to -53 and contains sequences capable of binding to a thrombin-induced nuclear factor, denoted as TINF, as well as constitutively expressed protein(s). The TINF complex appears rapidly in nuclear extracts of EC treated with thrombin, as early as 20-40 min post-stimulation, is relatively long-lived, being maintained up to 7 h, and is absent by 24 h after treatment. The appearance of the TINF complex in the nucleus does not require de novo protein synthesis, which may indicate that it pre-exists in an inactive form in the cell and that thrombin either directly or indirectly causes its activation and/or translocation.
We show by
competition in EMSA that the interaction between the thrombin-induced
nuclear proteins and the B region of the PDGF B-chain promoter is
specific. Among a panel of nuclear factor oligomers representing the
consensus sequence binding sites for AP1, AP2, NFB, TFIID, CREB,
Sp1, or oligonucleotides flanking the B region and the B region itself,
only an excess of unlabeled oligonucleotide B and consensus sequence
Sp1 oligonucleotide were effective competitors. Moreover, recombinant
Sp1 protein bound to labeled B oligomer and yielded a band that
corresponded in electrophoretic mobility to band 1 from the profile
obtained using nuclear extract from thrombin-stimulated EC. When HeLa
cell nuclear extracts (used as a known source of Sp1) (39) were
incubated with B region oligomer, two distinct bands were observed that
had similar gel mobilities to two bands seen with thrombin-stimulated
EC nuclear extracts. These bands may correspond to the two Sp1
proteins, of 105 kDa and 95 kDa, that have been described by
others(39) .
TINF and Sp1 are both nuclear factors that have the capacity to bind to oligonucleotide sequences present in the B region of the PDGF B-chain gene yet appear to be distinct proteins. Using heat treatment of nuclear extracts, we inhibited Sp1 binding, as described by others (35) , but not TINF binding to the B region sequence. Depletion of Sp1 from thrombin-stimulated EC nuclear extracts with polyclonal Sp1 antibody prior to binding with B region oligomer, dramatically reduced the Sp1 bands 1 and 2 without significantly changing the TINF band in an EMSA. In a supershift assay, in which the same nuclear extracts were allowed to bind to the B region oligomer first and then allowed to react with Sp1 antiserum, we observed retarded mobility of the SP-1 bands without a change in TINF mobility.
The GC box is the classic high affinity Sp1-binding site(40) . However, this is not the nuclear factor's target sequence in the ThR region. In the ThR region the sequence CCACCC is present as overlapping, inverted repeats. Extensive reports exist in the literature that show Sp1 is capable of binding to a CACCC sequence(36, 37, 38, 41) , which is present in each of the inverted repeats of the ThR region of the PDGF B-chain gene. Although Sp1 is widely distributed through many cell types and species, binding sites for this strong transcriptional activator have been reported in the promoters of many cell-specific genes. Direct interactions between Sp1 and a variety of other binding proteins have been implicated in several regulated processes, including hormone activation(42) , regulation by sterols(43) , heat shock response(44) , and tissue-specific gene expression(45) . Sp1 has been shown to contain several independent transcriptional activation domains in addition to the zinc finger region located within the C terminus of the protein (46) . Regions outside of the DNA binding domain of Sp1 may be responsible for modulating transcriptional activity by allowing for its interaction with other factors in the transcription initiation complex, such as TINF. Sp1 sites have been identified near binding sites for other transcriptional activators, such as CTF/NF-1(47) , AP2(48) , OTF-1(49) , and, in fact, a functional interaction between Sp1 and AP-1 has been demonstrated(50) . Recently, Sanchez et al.(51) have shown cooperation of a sterol regulatory element-binding protein and Sp1 in regulating the transcription of the low density lipoprotein receptor gene.
We have demonstrated that the ThR segment within the B region of the PDGF B-chain gene is involved in the transcriptional response to thrombin. The conservation of the ThR region of the PDGF B-chain gene across different species (human(52) , feline(52) , and murine(53) ) suggests that the region may serve as a binding site for important cis-acting elements. If this region is in fact a site responsive to a thrombin-induced transcriptional activator, then one might anticipate finding the CCACCC motif within the promoter region of other thrombin-responsive genes. In fact, the CCACCC-motif is also present in the promoters of other EC genes that are regulated by thrombin. The PDGF A-chain gene, which is induced by thrombin, contains this motif both in the sense and ``antisense'' DNA strands (positions 269 and 233 bp from the TATA box, respectively). The CCCACC motif is also present in the promoters of other thrombin-regulated genes, such as tissue factor (as GGGTGG at 85 bp and CCCACC at 291 bp from the TATA box), thrombomodulin (CCCACC at 56 bp and GGGTGG at 246 bp from the TATA box), VCAM-1 (at 117 bp from the TATA box), and von Willebrand factor (at 403 bp from the TATA box). The PAI-1 gene, another EC gene that is induced by thrombin, contains a GGGTGG at positions 46 and 401 from the TATA box, and it has been shown that the sequence GGGTGG in the sense strand at position 46 is responsible for TPA induction of this gene(50) .
Our studies with mutant sequence oligomers of the ThR region suggest that in thrombin-stimulated EC the CACCC motif of the PDGF B-chain promoter is the recognition site for both TINF and Sp1, and the orientation is of no consequence to nuclear factor binding. The mechanism by which these two nuclear factors mediate induction of transcription by thrombin is not known. In gel shift assays, we observed a reduction of Sp1 binding to the B region oligomer in thrombin-treated nuclear extracts, possibly because TINF and Sp1 bind in a mutually exclusive fashion and therefore compete for the oligomer.
Jin et al.(28) have recently identified a site in the PDGF B-chain promoter that was essential for phorbol ester-induced PDGF B-chain transcription in K-562 cells. Subsequent in vivo footprinting analyses by Dirks et al.(54) have confirmed that this region binds a transcriptional activator in phorbol ester-treated K562 cells. This reported TPA response element coincides with the region that we report as the ThR region. Our evidence indicates that TINF is a different nuclear factor than the TPA-induced protein identified by Jin et al. and referred to as band ``m''(28) . TINF has substantially greater gel mobility than band m and, unlike band m, appearance of TINF in the nucleus is independent of de novo protein synthesis. It should be noted that there is a difference in the numbering of the position of the ThR region in our report and the PMA-responsive region in the report of Jin and co-workers, which is based on a discrepancy in the literature of the position of the transcription initiation site of the PDGF B-chain gene. We have followed the mRNA initiation site described by Pech et al.(24) rather than that of Rao et al.(23) .
In conclusion, we have identified a sequence in the PDGF B-chain promoter which is involved in thrombin-induced transcription of this gene in bovine aortic EC. Mutational analysis of the ThR region has revealed that the CACCC-motif is required for a thrombin response. We also provide evidence of a specific thrombin-induced nuclear factor that binds to this region. Sp1 protein is also capable of binding to the CACCC sequence, but is distinct from TINF. The mechanism by which these transcription factors interact to regulate transcription of the PDGF B-chain gene is the subject of further investigation.