© 1999 American Heart Association, Inc.
Molecular Medicine |
Transcriptional Activation of the Zinc Finger Transcription Factor BTEB2 Gene by Egr-1 Through Mitogen-Activated Protein Kinase Pathways in Vascular Smooth Muscle Cells
From the Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, Japan.
Correspondence to Ryozo Nagai, MD, Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15, Showa-machi, Maebashi, Gunma, 371-8511, Japan.
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Abstract—We have recently demonstrated that a developmentally regulated zinc finger protein, basic transcription regulatory element binding protein 2 (BTEB2), is induced in neointimal smooth muscle in response to vascular injury. In this study, we investigated the molecular mechanisms regulating BTEB2 expression in vascular smooth muscle cells (SMCs) in vitro. BTEB2 mRNA expression is rapidly and persistently induced in SMCs by phorbol 12-myristate 13-acetate (PMA) and basic fibroblast growth factor. We have isolated and characterized the promoter region of the human BTEB2 gene to determine the regulatory network controlling expression of this gene in vascular SMCs. Functional studies on the BTEB2 promoter coupled to a luciferase reporter gene demonstrated activation of the promoter by PMA and basic fibroblast growth factor. Both characterization of DNA-protein complexes in vitro and site-specific mutation analysis of the BTEB2 promoter have defined a 9-bp sequence, 5'-CGCCCGCGC-3', located at -25, as the Egr-1 binding site mediating an induction of the BTEB2 promoter activity by PMA. In addition, we show that this site mediates inducible expression through the mitogen-activated protein kinase pathways. These results indicate that BTEB2 is a target of the early-response gene Egr-1, and mitogen-activated protein kinase pathways directly or indirectly activate BTEB2 expression. Given a rapid induction of Egr-1 on stimulation with growth factors or injury, these findings may represent at least one of the molecular mechanisms underlying phenotypic modulation of smooth muscles after vascular injury.
Key Words: BTEB2 • Egr-1 • mitogen-activated protein kinase • smooth muscle cell • phenotypic modulation
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Introduction |
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Understanding the molecular mechanisms underlying the diversity and plasticity of phenotypes displayed in vascular smooth muscle cells (SMCs) is of particular importance to elucidate the ontogenity of vascular lesions. It is well established that a number of genes important for the pathogenesis of vascular diseases are overexpressed or underexpressed in neointimal SMCs.1 2 These include smooth muscle myosin heavy chain, SM22
, caldesmon, and calponin. Because the genes for these proteins
are differentially expressed depending on the proliferative state of
SMCs, transcription factors of which the activity is regulated by
growth stimulation are responsible at least in part for the distinct
pattern of gene expression seen in neointimal SMCs. Studies over the last several years have identified transcription factors that are considered to be responsible for changes in the expression of a battery of genes in vascular lesions. The zinc finger transcription factor Egr-1 has been implicated in the activation of a number of pathophysiologically important genes,3 including transforming growth factor-ß1,4 tissue factor,5 urokinase-type plasminogen activator, and platelet-derived growth factor (PDGF)–A and -B.6 A recent study has demonstrated that Ets-1 is expressed in neointimal SMCs after balloon injury and in proliferating SMCs in vitro7 and is postulated to regulate the matrix metalloproteinase gene promoter.8 MEF2 has also been shown to be expressed at higher levels in proliferating SMCs than in differentiated SMCs.9 In contrast, a homeobox gene Gax10 11 and the members of the GATA family12 13 have been shown to promote growth arrest.
We have previously shown that the nonmuscle-type myosin heavy chain (SMemb/NMHC-B) gene is inducible in neointima14 15 and its induction is regulated by the zinc finger transcription factor basic transcription regulatory element binding protein 2 (BTEB2).16 17 BTEB2 contains 3 C2H2 zinc finger domains and belongs to a family of Krüppel-like zinc finger factors that include Sp1,18 19 GKLF,20 21 and LKLF.22 Although BTEB2 has initially been cloned from human placenta as a factor similar to BTEB,23 BTEB2 expression is found to be largely restricted to smooth muscle tissues and lung and down-regulated during aortic development. In addition, BTEB2 expression is observed in proliferating SMCs in response to balloon injury.17 From these studies, we hypothesize that BTEB2 may contribute to phenotypic modulation of vascular SMCs. However, the molecular basis for inducible expression of the BTEB2 gene in response to vascular injury remains to be determined.
In this article, we describe the isolation and characterization of a genomic clone containing the 5'-flanking region of the BTEB2 gene. The BTEB2 promoter-luciferase reporter genes were transfected into vascular SMCs, and cis-acting elements as well as trans-acting factors regulating promoter activity were analyzed. This study demonstrates that BTEB2 expression is induced through mitogen-activated protein (MAP) kinase pathways and directly regulated by the early-response gene Egr-1. Our results represent one of the molecular mechanisms by which growth stimulation leads to the phenotypic modulation of vascular SMCs.
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Materials and Methods |
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RNA Isolation and Northern Blot Analysis
Rabbit aorta-derived SMCs C2/2 cells were previously described.24 Total RNA was isolated from C2/2 cells treated with or without phorbol 12-myristate 13-acetate (PMA) (100 ng/mL) and basic fibroblast growth factor (bFGF; 10 ng/mL) using the ISOGEN (guanidinium isothiocyanate/phenol-chloroform) reagent in accordance with the manufacturer's instruction. RNA samples (15 µg) were separated on 1.2% formamide/agarose gels, transferred to a nylon membrane (Pall Biodyne), and then hybridized with either BTEB2 or Egr-1 cDNA as previously described.17
Western Blot Analysis and Anti-BTEB2 Antibody
Cytosolic extract from C2/2 cells stimulated with 100 ng/mL PMA
for 6 hours was electrophoresed on 15% SDS-polyacrylamide gels
and transferred onto nitrocellulose membrane (Schleicher and Schuell,
Inc). Egr-1 and BTEB2 proteins were detected according to the enhanced
chemiluminescence protocol (Amersham Corp) using a 1:1000 dilution of a
rabbit anti–Egr-1 antiserum (Santa Cruz Biotechnology, Inc; 100 µg
IgG/mL) or 1:100 dilution mouse monoclonal anti-BTEB2 antibody.
Production of anti-BTEB2 antibody has been
described.17
Isolation and Sequencing Analysis of the Human BTEB2
Gene
A human placenta genomic library (Stratagene) was screened with
a 657-bp cDNA probe containing the full length of the coding region of
rabbit BTEB2 cDNA.23 After 3 rounds of screening, 4
positive clones were isolated. One of these clones was selected for
restriction enzyme and Southern blot analysis. The DNA fragment
containing the 5' end of the BTEB2 gene was identified by hybridizing
the membranes with a 290-bp human BTEB2 cDNA probe containing the
published 5'-untranslated region.23 A 6.6-kb
XbaI fragment was subcloned into pBluescript II SK(-)
(Stratagene), and the resultant plasmid was used for further
restriction enzyme mapping and nucleotide sequencing.
Sequencing was performed using the cycle sequencing method with
SequiTherm EXCEL II DNA Polymerase (SequiTherm EXCEL Long-Read DNA
Sequence Kit-LC, Epi-Centre Technologies) in a LI-COR model 4000L
automated DNA sequencer according to the manufacturer's instructions.
Both strands of DNA were sequenced. Sequence analysis was
performed using Gene Works software (Oxford Molecular Group, Inc).
S1 Nuclease Mapping
A 219-residue oligonucleotide complementary to
-163 to +56 with respect to the transcription start site was
synthesized by polymerase chain reaction (PCR), and the 5' end was
labeled with [
-32P]ATP and T4
polynucleotide. This primer (1x104
cpm) was then hybridized to 1 µg mRNA of NCI-H322 cells (human
bronchoalveolar carcinoma cells) at 42°C for 36 hours in
hybridization buffer (containing 40% deionized formamide and, in
mmol/L, sodium citrate [pH 6.4] 50, sodium acetate [pH 6.4[ 150,
and EDTA 0.5). After S1 nuclease treatment, protected fragments were
analyzed in an 8 mol/L urea/8% polyacrylamide gel.
Promoter-Luciferase Vector Chimeric Construct
The BTEB2 promoter-luciferase reporter genes were constructed by
cloning a XbaI-SacII fragment that corresponds to
-2300 to +236 relative to the transcriptional start site into pGVB
(PicaGene). Serial deletion constructs were prepared as follows:
ApaI-SacII fragment (-363 to +236) and
SacI-SacII (-67 to +236) fragments were cloned
into the corresponding sites of pGVB, and resultant plasmids were
designated as -363Luc and -67Luc, respectively. The forward and
reverse primers used for generation of -32Luc were
5'-GTGCGCCCGCCCGCGCCTGGA-3' and 5'-TTTAGCTTCC-GGTGGCGGCG
GGCTGGGCG-3'. The forward and reverse primers used for generation of
-388Luc were 5'-CCCGGTACC-CGGCGCTGCCAATCAGGCGAT-3' and
5'-TTTAAGCTTCCG-GTGGCGGCGGGCTGGGCG-3', with
KpnI and HindIII sites (underlined),
respectively.
For generation of site-directed mutants within the GC-1 in the BTEB2 promoter, recombinant PCR with 2 rounds of amplification was performed in the context of the -388Luc construct. The PCR primers (mutations of wild-type sequence appear in boldface) for -388 mut1Luc were 5'-GTGCATTCGCCCGCGCCTGGACTG-3' (sense) and 5'-TTTAAGCTTCCGGTGGCGGCGGGCTGGGCG-3'(antisense). In brief, sense and antisense primers with the corresponding mutations were synthesized and incubated in separate reaction tubes with -2300Luc as template, upstream primer (nucleotide -388), and reverse primer (nucleotide +236), thus yielding 2 subfragments that each contained the appropriate mutation. Subfragments were gel purified and annealed, and a second round of PCR was performed using upstream primer (nucleotide -388) and reverse primer (nucleotide +236). The PCR products were then isolated and subcloned into the KpnI/HindIII sites of pGVB, as above. Resultant plasmid was designated as -388 mut1Luc. All constructs were verified by sequencing the inserts and flanking region in the plasmid. The reporter plasmids of -388 mut2Luc, -388 mut3Luc, and -388 mut4Luc were constructed in the same ways. The PCR primers (mutations of wild-type sequence appear in boldface) for -388 mut2Luc, -388 mut3Luc, and -388 mut4Luc were as follows: -388 mut2Luc, 5'-GTGCGCCTATCCGCGCC-TGGACTG-3'; -388 mut3Luc 5'-GTGCGCCCGCTTACGCCTGGACTG-3'; and -388 mut4Luc 5'-GTGCGCCCGCCCGTATCTGGACTG-3'.
Expression Plasmid
For generation of the Egr-1 expression plasmid, the coding
region of mouse Egr-1 cDNA (obtained from American Type Culture
Collection) was amplified by PCR using the upstream primer with an
EcoRI site (underlined),
5'-GGGGAATTCTCCAGCTCGCTGGT-CCGGCAT-3', and the reverse
primer with an XbaI site (underlined),
5'-GGGTCTAGACCTTTAGCAAATTTCAATTGT-3'. The PCR product
was gel-purified, digested, and subcloned into the
EcoRI/XbaI site of pcDNA3 (Invitrogen). The
expression plasmid MAP/ERK kinase 1/cytomegalovirus (MEK1/CMV) and the
MEK1 inactive form/CMV (kindly provided by Roger J. Davis, Howard
Hughes Medical Institute, Worcester, MA) has been
described.25
Transfection and Luciferase Assay
For transfections, C2/2 cells were plated on 35-mm culture
dishes (Falcon) at a density of 104 cells/dish
and incubated in DMEM supplemented with 10% FBS for 24 hours. Each
dish was transfected at 70% confluency with 1 µg of a BTEB2
promoter-luciferase fusion plasmid. Gene transfections were performed
by a modified calcium phosphate precipitation method as previously
described.17 Transfections were typically carried out for
12 to 16 hours followed by 48 hours of recovery and growth. Cells were
harvested in cell lysis buffer. Luciferase activities were assayed
according to the manufacturer's specifications (Promega). A Berthold
Lumat LB 9507 luminometer was used to measure the light reactivity of
firefly luciferase. The data are mean±SEM of at least 4 independent
experiments in duplicate.
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift
Assay (EMSA)
Nuclear extracts from C2/2 cells were prepared as previously
described.26 Synthetic double-strand
oligonucleotides used in gel mobility shift assays were
-32P-labeled at the 5' end using T4
polynucleotide kinase. Binding reactions contained 20 µg
of nuclear extracts, 1 µg of poly(dI-dC) as a nonspecific competitor
in nuclear lysis buffer, 
10 000 cpm labeled probe, and 100-fold
molar excess of competitor oligonucleotide where
appropriate. Protein binding was allowed to proceed for 30 minutes at
room temperature and then analyzed on a 5%
polyacrylamide gel at 100 V, and gels were dried and exposed to
Kodak XR film. For experiments using antibodies, 1 µL of anti–Egr-1
or Sp1 polyclonal antibodies (Santa Cruz Biotechnology, Inc; 100 µg
IgG/0.1 mL) was added to the protein extract plus buffer, and 30-minute
preincubation on ice was carried out before the addition of
radiolabeled oligonucleotide. Nucleotide
sequences of the oligonucleotides used for EMSA were as
follows: GC-1, 5'-GTGCGCCCGCCCGCGCCTGGA-3'; mut1,
5'-GTG-CATTCGCCCGCGCCTGGA-3'; mut2,
5'-GTGCGCCTAT CCGCGCCTGGA-3'; mut3,
5'-GTGCGCCCGCTTACGCCTGGA-3'; mut4,
5'-GTGCGCCCGCCCGTATCTGGA-3'; Sp1,
5'-AATC-GATCGGGGCGGGGCGAGC-3'; Egr-1,
5'-CCCGGCGCGGGGG-CGATTTCGAGT-3'; and Ets-1,
5'-GAGCACAGTCGAGGAAG- TGACTAACTG-3'. Mutations of
wild-type sequence appear in boldface. Consensus binding sites for
Egr-1, Sp1, and Ets-1 are underlined.
Immunohistochemistry
Vascular injury was produced by endothelial cell
denudation of the aorta using a balloon catheter in 3 Wistar rats (8
weeks old). Rats were euthanized at 2 weeks after injury. Aorta was
fixed in 10% formalin and paraffin-embedded. Sections were stained
with affinity-purified rabbit IgG against BTEB2, Sp1 (PEP2, Santa Cruz
Biotechnology, Inc) and Egr-1 (C-19, Santa Cruz Biotechnology, Inc) by
using Vectastain Elite ABC kit (Vector Laboratories) as previously
described.17 This investigation conforms to the guide for
the care and use of laboratory animals approved by the committee
of the Gunma University School of Medicine.
Statistical Analysis
Data are reported as mean±SEM. The difference between means was
evaluated using the Student t test. Significance levels were
established at P<0.05.
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Results |
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PMA and bFGF Induce BTEB2 mRNA in Cultured Vascular SMCs
Our previous immunohistochemical studies showed that BTEB2 expression is increased in neointimal SMCs after balloon injury of rat aorta.17 In this report, we set out to determine the effects of mitogenic stimulation on BTEB2 expression in vascular SMCs. We used C2/2 cells, a rabbit aorta-derived SMC line, in this study. C2/2 cells grown in the presence of either PMA (100 ng/mL) or bFGF (10 ng/mL) showed a significant increase in the uptake of 3H-labeled methylthymidine (data not shown), thus indicating that either PMA or bFGF exerts its mitogenic effects on C2/2 cells. We then examined the effects of these mitogens on the BTEB2 mRNA and protein levels. Northern blot analysis showed that BTEB2 transcript is present in untreated cells, and both PMA and bFGF increased the BTEB2 mRNA levels 2.5-fold and 2.1-fold over vehicle, respectively (P<0.05, n=4) (Figure 1
).
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Isolation of Genomic Clones and Sequence Analysis
To determine the molecular mechanisms by which mitogens increase
the expression of BTEB2, we have isolated and characterized the human
BTEB2 gene. A human genomic library was screened with a
32P-labeled human BTEB2 cDNA probe under
conditions of high stringency. A screen of 1x106
independent plaques yielded 4 positive recombinant phages. They were
subjected to further analysis by restriction enzyme mapping and
Southern blotting. One of these clones, 
hBTEB2-1, carried a DNA
insert of 20 kb and encoded the 5'-flanking region and the first and
the second exons.
In this study, we focused our efforts on the characterization of the
5'-flanking region of the BTEB2 gene, a region thought to contain the
putative promoter and regulatory sequences. A 6.6-kb XbaI
fragment from the clone BTEB2-1, which hybridized with the
5'-untranslated region of the human BTEB2 cDNA, was subcloned into
pBluescript SK(–) vector for sequence analysis. Figure 2A shows a partial restriction map of the
6.6-kb XbaI DNA fragment and its nucleotide
sequence comprising exons 1 and 2. Exon 1 (263 bp long) contains only
the 5'-untranslated sequence. The exon/intron boundaries for exons 1
and 2 in the BTEB2 gene were determined by comparing genomic DNA
sequences with the published cDNA sequence.23
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Location of the Transcription Initiation Site
To identify the transcription start site for the BTEB2 mRNA, S1
nuclease mapping was performed. We used human bronchoalveolar cell
carcinoma NCI-H322 cells, which express BTEB2 mRNA (data not shown), to
obtain human BTEB2 mRNA. Poly (A)+ RNA from these
cells was hybridized with 5'-end-labeled antisense single-stranded DNA
probes (Figure 2C
), as described in Materials and Methods. The
signal was detected at 56 bp long as a protected band. The
nucleotide sequence of 928 bp located upstream from the
transcription initiation site of the BTEB2 gene is shown in Figure 2B. Neither a canonical TATA box nor CAAT box was found in this
promoter. The motifs of known cis-regulatory elements were
searched by transcription factor databases (Transfec Matrix Table). The
putative Sp1 binding site 5'-GGCGGG-3', present in constitutively
expressed genes, was found 8 times, at -419, -362, -333, -262,
-224, -219, -45, and -27. A GATA box sequence,
5'-(A/T)GATA(A/G)-3', which has been implicated in tissue-specific
expression of genes in skeletal and cardiac muscle, was found at -852
and -605. A consensus sequence for the binding of Egr-1, a zinc finger
transcription factor that may function as a growth-regulatory
transcription factor, was found at -27.
Definition of the PMA Response Element Within the BTEB2
Promoter
To determine whether PMA and bFGF increase BTEB2 mRNA levels at
the transcription level, -2300Luc, a luciferase reporter construct
containing a DNA segment extending from -2300 bp to +236 bp coupled to
a luciferase gene, was transiently transfected into C2/2 cells, treated
with PMA or left untreated, and assayed for luciferase activity (Figure 3A). PMA stimulated luciferase activity
of this construct 9-fold. We obtained the same result in primary
culture of rat SMCs (data not shown). PMA-induced activity was observed
in -363Luc, -67Luc, and -32Luc, of which the 5' ends correspond to
-363, -67, and -32 from the transcription start site, respectively.
All of these constructs were able to respond to PMA with an effective
range of 11- to 20-fold, whereas promoterless construct pGVB was
unresponsive. These results indicate that the sequence between -32 and
+230, to which we refer as the minimal promoter region, is sufficient
for PMA-induced activation of BTEB2 promoter. Furthermore, to delineate
the sequences within the BTEB2 promoter that are necessary for inducing
promoter activity in response to PMA, substitutions of 3
successive bases were made in the luciferase reporter plasmid -388Luc
(Figure 3B and 3C). The luciferase activity in cells transfected
with these base substitution mutants revealed that 9 bases encompassed
by mut2, mut3, and mut4 were necessary to show an induction in response
to PMA. The 9-base sequence, 5'-CGCCCGCGC-3', lying between -25 and
-17, perfectly matches the consensus Egr-1 site,
5'-CGCCC(C/G/T)CGC-3'.27 28
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Identification of Nuclear Factor Binding Sites Within the Minimal
Promoter Region
To better characterize the PMA-inducible cis-acting
element, we examined the binding of nuclear proteins extracted from
unstimulated and PMA-stimulated C2/2 cells to the region of the BTEB2
minimal promoter by EMSA. Two oligonucleotides encoding
the 5' and 3' portions of this region were synthesized and termed GC-1
and GC-2, respectively. Each oligonucleotide was 24 bp
long and contained a 9-bp overlapping stretch at their ends.
Double-stranded oligonucleotides GC-1 but not GC-2
formed 2 shifted complexes with nuclear extracts from either untreated
or PMA-treated C2/2 cells (Figure 4A).
The intensity of the DNA:protein complex with slower mobility (C1) but
not with faster mobility (C2) was increased in nuclear extracts from
PMA-treated cells. Addition of a 100-fold excess of unlabeled mutated
probes (mut1) abolished complex C1, whereas the
oligonucleotides, mut2, mut3, and mut4, had little or
no effect on complex formation. Formation of complex C1 was also
competed by a consensus sequence for the Egr-1 binding site but not for
the Sp1 and Est-1 binding sites (Figure 4B). In contrast,
formation of complex C2 was reduced by each of the unlabeled
competitors, thus suggesting that C2 is a nonspecific band. To further
identify the nuclear proteins present in complex C1, supershift
experiments were performed. Incubation of the nuclear extracts with
anti–Egr-1 antibody clearly supershifted complex C1. Anti-Sp1 antibody
had no effect on complex C1 formation (Figure 4C). These results
indicate that Egr-1 constitutes the major component of complex C1.
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PMA Stimulates Egr-1 and BTEB2 Expression With Different Kinetics
in C2/2 Cells
To investigate whether increased binding of Egr-1 to the BTEB2
promoter is accompanied by elevated cellular levels of Egr-1, we
analyzed basal and PMA-stimulated Egr-1 expression in C2/2
cells by Northern and Western blot analyses. PMA increased
Egr-1 mRNA and protein amounts, although a noticeable amount of Egr-1
protein was expressed before PMA stimulation (Figure 5A). As described in a variety of cell
types, Egr-1 mRNA is rapidly and transiently induced by PMA in C2/2
cells. In contrast, BTEB2 mRNA levels were gradually increased, with a
peak at 4 hours after stimulation. Consistently, Western blot
analysis showed an increase in BTEB2 and Egr-1 protein by PMA
stimulation (Figure 5B). It is noteworthy that anti-BTEB2
antibody detected 2 distinct bands in whole-cell lysates of C2/2, with
approximate molecular masses of 35 and 40 kDa.
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Overexpression of Egr-1 Stimulates BTEB2 Promoter
To demonstrate that elevation of cellular Egr-1 levels has
functional effects on the transcriptional activity of the BTEB2
promoter, we transiently overexpressed Egr-1 by transfection of the
Egr-1 expression vector Egr-1/pcDNA3 into C2/2 cells (Figure 6A) and rat SMCs. Cells transfected with
either -363Luc, -67Luc, or -32Luc expressed 8-fold greater
luciferase activity in the presence of Egr-1/pcDNA3 compared with the
vector pcDNA3. Cells transfected with -388Luc (wild type) and -388
mut1Luc expressed 5- to 6-fold greater luciferase activity in the
presence of Egr-1 expression vector compared with the vector pcDNA3. In
contrast, luciferase activity was not increased in cells transfected
with -388 mut2Luc, -388 mut3Luc, or -388 mut4Luc, which carries a
mutation within the Egr-1 site (Figure 6B). Because mutations
encompassed by mut2, mut3, and mut4 abolished binding of Egr-1 (Figure 4C), these results correlate Egr-1 binding to the BTEB2 promoter
with its transcriptional activation.
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MAP Kinase Pathway Activates BTEB2 Promoter
Mitogenic stimulation of vascular SMCs
activates downstream signaling pathways, such as MAP kinase
pathways.29 30 31 To determine the effects of MAP kinase
activation on BTEB2 expression, we examined PMA-stimulated BTEB2 mRNA
levels in C2/2 cells in the presence of MAP kinase
inhibitors. As shown in Figure 7A, PD98059 (50 µmol/L, a specific
inhibitor for MEK1, a MAP kinase kinase for extracellular
signal–regulated protein kinase [ERK]) but not SB203580 (10
µmol/L, a specific inhibitor for p38) blocked PMA-induced
BTEB2 expression. These results suggest that BTEB2 is activated
by PMA through MEK1 but not through p38. To determine the effects of
MEK1 on BTEB2 expression, C2/2 cells were transfected with expression
vector for MEK1 or empty vector pRc/CMV, along with several 5'-deletion
mutants of BTEB2-luciferase reporter gene. Expression of MEK1
stimulated the luciferase activity of -2300Luc, -363Luc, and -67Luc
by 8.4-fold, 17.2-fold, and 12.3-fold, respectively (Figure 7B).
To localize the sequence that is necessary for MEK1-mediated induction
of BTEB2 promoter, site-specific mutation constructs were used.
Induction of luciferase activity by MEK1 expression vector was
significantly reduced by introducing a mutation into the Egr-1 binding
site spanning between -25 and -17 in the context of -388Luc (Figure 7C). Consistent with these results, forced
expression of MEK1 stimulated the luciferase activity of -2300Luc. In
contrast, inactive mutant failed to activate the promoter
(Figure 7D). These results demonstrate that activation of MEK1
is sufficient to stimulate BTEB2 promoter activity and that the Egr-1
binding site mediates this effect.
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BTEB2 and Egr-1 Expression in Neointimal SMCs
To examine the induction of BTEB2 and Egr-1 in injured arteries,
denudation of rat aorta by a balloon catheter was performed, and
control and injured aorta at 2 weeks after injury were examined for
expression of BTEB2, Egr-1, and Sp1 by immunohistochemistry (Figure 8). Positive staining for BTEB2 and Egr-1
was observed in the neointimal layer of the injured aorta.
In the medial smooth muscle, only a few cells underneath the internal
elastic lamina were positive for BTEB2 and Egr-1. Neither BTEB2 nor
Egr-1 was detected in control aorta. In contrast, Sp1 was
constitutively expressed in neointimal and medial layers in
both control and injured aortas.
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Discussion |
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Our previous studies indicate that the zinc finger transcription factor BTEB2 regulates the SMemb/NMHC-B gene.17 BTEB2 is expressed in fetal aorta but is scarcely expressed in adult aorta, and its expression is reinduced in neointimal SMCs in response to balloon injury. In this study, we investigated the molecular mechanisms of the regulated expression of BTEB2 in vascular SMCs. Molecular cloning and sequence analysis of the human BTEB2 promoter revealed common features of a TATA-less promoter; multiple GC boxes were found in the proximal promoter region. Functional and DNA binding analyses defined Egr-1 consensus sequence lying between -25 and -17 as essential motif for PMA-induced BTEB2 promoter activation. In addition, our data indicate that the Egr-1 binding site is capable of mediating the effect of the MAP kinase pathway, suggesting that growth stimulation activates BTEB2 gene expression, which in turn promotes the phenotypic modulation of vascular SMCs.
One of the main conclusions in this study is that Egr-1 activates the BTEB2 promoter. Khachigian et al4 have recently reported that Egr-1 plays a role in inducing the expression of various genes during vascular injury. These studies suggest that Egr-1 binds to a cryptic element overlapping the Sp1 site in the PDGF-B promoter, displaces the prebound Sp1, and occupies the GC box on denudation of endothelium. The same mechanisms have been proposed for expression of shear stress–induced tissue factor, PDGF-A gene, and PMA-stimulated tissue factor gene.5 32 Although the Egr-1 site is embedded in a G+C-rich sequence (GC-1 sequence) lying at -32 of the BTEB2 promoter, Sp1 does not seem to bind to the GC-1 sequence.
Several observations indicate that the ERK/MAP kinase pathway activates the BTEB2 promoter. First, expression of constitutively active MEK1, a MAP kinase kinase that phosphorylates ERK,33 stimulated BTEB2 promoter activity. This activation is considered to be specific because inactive mutant failed to activate the promoter, and MEK1 was not capable of activating the promoter whose Egr-1 binding site is disrupted. Second, both PMA and bFGF are known to activate the ERK pathway rather than JNK/SAPK or p38 in a variety of cell types, including vascular SMCs.29 30 31 Third, PD98059 (a specific inhibitor for MEK134 ) but not SB203580 (a specific inhibitor for p3835 ) blocks PMA-induced BTEB2 expression. Given that MAP kinase is the essential part of the signal transduction machinery and occupies a central position in cell growth,36 transcriptional activation of the BTEB2 gene by the MAP kinase pathway supports our notion that BTEB2 plays an important role in the phenotypic modulation of neointimal SMCs. Results of transient transfection experiments using primary cultures of rat aortic SMCs in place of C2/2 cells conformed to this hypothesis (data not shown).
Our results suggest that the Egr-1 site is involved in MEK1-mediated activation of the BTEB2 promoter. This finding is of particular interest, because transcriptional regulation by MEK-ERK pathways typically involves transcription factors such as the ternary complex of the serum response factor–ternary complex factor, which binds to the serum response elements (SREs). Inducible expression of Egr-1 is known to involve cooperative interaction between serum response factor and ternary complex factors at SREs within the Egr-1 gene promoter.37 38 These considerations led us to suggest that activation of MEK1 induces Egr-1 through the SRE sites that in turn binds to Egr-1 binding site within the BTEB2 promoter and activates BTEB2 transcription. In fact, we found that the quantity of Egr-1 that binds to DNA is increased in response to PMA stimulation.
Our study showed that induction of BTEB2 mRNA is as rapid as that of Egr-1 mRNA. Accordingly, it is likely that the MEK1-ERK pathway directly phosphorylates preexisting Egr-1 and potentiates its transactivating function or DNA binding activity. There are precedent reports that suggest posttranscriptional modification of Egr-1.39 40 These studies demonstrate that DNA binding activity of Egr-1 is regulated by phosphorylation/dephosphorylation, given that the binding of Egr-1 to its consensus sequence is strongly but transiently stimulated by the phorbol ester or protein phosphatase inhibitor okadaic acid.39 41 42 Further studies are necessary to address this important question.
The identification of the Egr-1 binding site as a PMA response element in the BTEB2 gene will expand our knowledge concerning the mechanisms behind phenotypic modulation occurring in response to vascular injury. Previous studies from our laboratory and others demonstrated that BTEB2 binds to the G+C–rich sequence that is indistinguishable from the Sp1 binding site.17 23 Furthermore, we have recently found that BTEB2 is capable of activating transcription of many genes that are implicated in cellular proliferation, phenotypic modulation of SMCs, and thrombosis (K.K.-K., M.K., R.N., unpublished data, 1999). Injury-induced BTEB2 and Egr-1 expression is both spatially and temporally consistent with a possible role for these 2 zinc finger proteins in the regulation of multiples genes involved in vascular disease. The coordinate expression of growth factors and components of the coagulation system mediated by BTEB2 and Egr-1 may contribute to the complex series of cellular and thrombogenic events in the development of vascular occlusive lesions.
In conclusion, the functional data taken together with the DNA binding data support the hypothesis that the response of the BTEB2 promoter to PMA is mediated by Egr-1. Taking into account that the time course of the induction of BTEB2 by PMA is long lasting compared with that of Egr-1, the findings in the present study may represent one of the molecular mechanisms underlying sustained activation of many genes in phenotypically modulated SMC subsequent to the activation of early-response genes, including Egr-1.
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Acknowledgments |
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This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and a grant from the Japan Cardiovascular Foundation (to M.K. and R.N.). We thank Dr T. Suzuki for assistance in preparation of the manuscript.
Received May 11, 1999; accepted August 19, 1999.
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