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Circulation Research. 1999;85:787-795

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(Circulation Research. 1999;85:787-795.)
© 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

Keiko Kawai-Kowase, Masahiko Kurabayashi, Yoichi Hoshino, Yoshio Ohyama, Ryozo Nagai

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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{alpha}, 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.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 [{gamma}-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 {gamma}-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, {approx}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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 1Down).



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  		Figure 1. Effects of PMA and bFGF on BTEB2 mRNA levels. Top, Northern blot analysis of RNA prepared from cultured C2/2 cells treated with 100 ng/mL PMA and 10 ng/mL bFGF for 6 hours and hybridized to the radiolabeled rabbit BTEB2 cDNA probe. 28S ribosomal RNA stained with methylene blue indicates that comparable amounts of total RNA were blotted onto a membrane. Bottom, Quantification of BTEB2 mRNA expression normalized to the intensity of 28S rRNA. Values are mean±SEM. An arbitrary value of 1.0 was assigned to vehicle-treated cells (cont).

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, {lambda}hBTEB2-1, carried a DNA insert of {approx}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 {lambda}BTEB2-1, which hybridized with the 5'-untranslated region of the human BTEB2 cDNA, was subcloned into pBluescript SK(–) vector for sequence analysis. Figure 2ADown 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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  		Figure 2. Sequence of the 5'-flanking region and exon 1 of the human BTEB2 gene. A, Structure and restriction map of the BTEB2 genomic clone containing the 5'-flanking region, exon 1, exon 2, and intron 1. Open boxes indicate exons 1 and 2. Restriction enzyme sites are indicated as follows: X, XbaI; A, ApaI; SI, SacI; SII, SacII; and K, KpnI. B, Nucleotide sequence of the 5'-flanking region and exon 1 of the human BTEB2 gene. The transcription start site identified by S1 nuclease mapping is designated as +1. Nucleotides are numbered on the left. Entire exon 1 sequence is shown in rectangle. Sp1 site and GATA box are underlined. Egr-1 site is boxed. C, S1 mapping of the transcription start site. Top, Schematic representation of the S1 probe, 219-bp fragment containing the putative transcription start site. Exon 1 is boxed. Restriction enzyme sites are indicated as follows: A, ApaI; SI, SacI; and SII, SacII. Bottom, End-labeled S1 probe was incubated with 1 µg of mRNA from NCI-H322 cells, digested with 250 units of S1 nuclease, and then subjected to electrophoresis on an 8 mol/L urea/8% polyacrylamide gel. Sequence ladder of M13 DNA was used as a size marker. Protected band is indicated by arrow.

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 2CUp), 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 2BUp. 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 3ADown). PMA stimulated luciferase activity of this construct {approx}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 3BDown and 3CDown). 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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  		Figure 3. Effect of PMA on the BTEB2 promoter activity. A, A series of constructs with the BTEB2 promoter of various lengths was transfected into C2/2 cells. Plasmids -2300Luc, -363Luc, -67Luc, and -32Luc contain the BTEB2 promoter region up to -2300, -363, -67 and -32, respectively. The 3' end of the promoter of all of these constructs corresponds to +236. Transfected cells were incubated with vehicle or PMA (100 ng/mL) for 24 hours as described in Materials and Methods. B, Schematic representation of the base substitutions introduced into -388Luc. Mutations of wild-type sequence appear in boldface. C, Luciferase activity associated with the base substitution shown in panel A. Cells transfected with indicated reporter genes were incubated with vehicle or PMA (100 ng/mL) for 24 hours as described in Materials and Methods. Luciferase activities are expressed relative to that of promoterless plasmid pGVB, which is set at 1.0. Values are mean±SEM. Rectangles indicate Sp1 site; ovals, Egr-1 site.

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 4ADown). 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 4BDown). 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 4CDown). These results indicate that Egr-1 constitutes the major component of complex C1.



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  		Figure 4. EMSA. A, Nuclear extracts from C2/2 cells grown in the absence or presence of PMA were incubated with GC-1 probe and subjected to electrophoresis on a 5% polyacrylamide gel. The major retarded complexes are denoted as C1 and C2. B, Competition assays were performed using the indicated unlabeled double-stranded oligonucleotides as competitors. Nucleotide sequence of the probe and competitors are described in Materials and Methods. C, Supershift assays were performed using the indicated antibodies. Supershifted complex is indicated by arrows.

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 5ADown). 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 5BDown). 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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  		Figure 5. Effects of PMA on BTEB2 and Egr-1 expression. A, Time course of the induction of Egr-1 and BTEB2 mRNAs by PMA. C2/2 cells were incubated with 100 ng/mL PMA for indicated times, and the steady-state mRNA levels for Egr-1 and BTEB2 were measured by Northern blot analysis. 28S ribosomal RNA stained by methylene blue indicates that comparable amounts of total RNA were blotted onto a membrane. B, Western blot analysis of BTEB2 and Egr-1 in C2/2 cells treated with PMA. Cytosolic extract from C2/2 cells stimulated with 100 ng/mL PMA for 6 hours was electrophoresed on 15% SDS-polyacrylamide gels, transferred onto a nitrocellulose membrane, and immunostained with either anti-BTEB2 or Egr-1 antibody.

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 6ADown) and rat SMCs. Cells transfected with either -363Luc, -67Luc, or -32Luc expressed {approx}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 {approx}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 6BDown). Because mutations encompassed by mut2, mut3, and mut4 abolished binding of Egr-1 (Figure 4CUp), these results correlate Egr-1 binding to the BTEB2 promoter with its transcriptional activation.



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  		Figure 6. Effects of mutation of Egr-1 binding site on the BTEB2 promoter activity. A, C2/2 cells were transfected with the indicated reporter plasmids along with either control vector (pcDNA3) or Egr-1 expression vector (Egr-1/pcDNA3). B, Luciferase activity associated with the base substitution of Egr-1 binding site. Indicated reporter genes were cotransfected with either pcDNA3 or Egr-1/pcDNA3. Luciferase activity is expressed relative to that of promoterless plasmid pGVB, which is set at 1.0. Values are mean±SEM.

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 7ADown, 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 7BDown). 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 7CDown). Consistent with these results, forced expression of MEK1 stimulated the luciferase activity of -2300Luc. In contrast, inactive mutant failed to activate the promoter (Figure 7DDown). 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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  		Figure 7. Effects of MEK1 expression vector on the BTEB2 promoter activity. A, C2/2 cells were pretreated with PD98059 (50 µmol/L) or SB203580 (10 µmol/L) for 1 hour and were exposed to PMA (100 ng/mL) for 4 hours. Total cellular RNA (20 µg) was analyzed by Northern blotting for BTEB2 mRNA. 28S ribosomal RNA stained by methylene blue indicates that comparable amounts of total RNA actually blotted onto a membrane. B, Effects of MEK1 expression on the 5' deletion constructs. C2/2 cells were transfected with the indicated reporter plasmids along with either control vector (pRc/CMV) or MEK1 expression vector (MEK1/CMV). C, Luciferase activity associated with the base substitution of Egr-1 binding site. Indicated reporter genes were cotransfected with either control vector (pRc/CMV) or MEK1 expression vector (MEK1/CMV). D, C2/2 were transfected with -2300Luc along with the expression vector for MEK1 or inactive mutant of MEK1. Luciferase activity is expressed relative to that of pGVB, which is set at 1.0. Values are mean±SEM.

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 8Down). 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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  		Figure 8. Immunohistochemistry for BTEB2, Sp1, and Egr-1 in rat aorta after balloon injury. Rat aorta 2 weeks after balloon injury was stained with antibodies against BTEB2, Sp1, and Egr-1. H&E denotes hematoxylin and eosin staining. Cells in neointima (i) were clearly positive for BTEB2 and Egr-1, but these proteins were scarcely expressed in cells in medial SMCs (m). Sp1 is constitutively expressed in both intimal and medial SMCs before and after injury. Cont indicates control; 2w, 2 weeks after injury.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *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.


*    Acknowledgments
 
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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 up arrowMaterials and Methods
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 *References
 

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