© 1999 American Heart Association, Inc.
Original Contributions |
BTEB2, a Krüppel-Like Transcription Factor, Regulates Expression of the SMemb/Nonmuscle Myosin Heavy Chain B (SMemb/NMHC-B) Gene
From The Second Department of Internal Medicine (N.W., M. Kurabayashi, Y.S., K.K.-K., Y.H., I.M., M.A., M. Kuro-o, R.N.), Gunma University School of Medicine, Gunma, and The Third Department of Internal Medicine (M.W., T.S., Y.Y.), University of Tokyo, Japan.
Correspondence to Ryozo Nagai, MD, The Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma, 375-8511, Japan. E-mail nagai{at}news.sb.gunma-u.ac.jp
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Abstract |
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We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a
gt11 cDNA library prepared from C2/2 cells, a
rabbit-derived SMC line, by using a radiolabeled concatenated
oligonucleotide containing SE1 as a probe. Sequence
analysis revealed that one of the cDNA clones corresponds to
the rabbit homologue of basic transcriptional element binding protein-2
(BTEB2), which has previously been identified as one of the
Krüppel-like transcription factor. Gel mobility shift assays and
antibody supershift analyses with nuclear extracts from C2/2
cells indicate that BTEB2 is a major component of nuclear factor:SE1
complexes. Furthermore, a glutathione S-transferase-BTEB2
fusion protein binds to the SE1 in a sequence-specific manner. In
support of the functionality of BTEB2 binding, basal promoter activity
and BTEB2-induced transcriptional activation were markedly attenuated
by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was
most highly expressed in intestine, urinary bladder, and uterus. BTEB2
mRNA levels were downregulated in rabbit aorta during normal
development. Moreover, immunohistochemical analysis indicated a
marked induction of BTEB2 protein in the neointimal SMC
after balloon injury in rat aorta. These results suggest that BTEB2
mediates the transcriptional regulation of the SMemb/NMHC-B gene and
possibly plays a role in regulating gene expression during phenotypic
modulation of vascular SMC.
Key Words: SMemb/NMHC-B • smooth muscle cell • basic transcriptional element binding protein-2
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Introduction |
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Proliferation and phenotypic modulation of vascular smooth muscle cells (SMCs) play a major role in the pathogenesis of cardiovascular diseases, including atherosclerosis and restenosis after balloon angioplasty.1 Phenotypic modulation is characterized by the loss of expression of the SMC-specific genes as well as a selective upregulation of fetal/neonatal isoforms of the contractile proteins, extracellular matrix proteins, growth factors, and their receptors.2 3 4 5 6 The mRNA population particular to either adult- or embryonic-phenotype of SMCs is determined in part by its characteristic array of transcription factors. Alteration of phenotype from embryonic/fetal type to adult type or vice versa may require a change in expression level of a member of these transcription factor subsets. Significant progress has been made in the identification and characterization of receptors for growth factors and cytokines responsible for the proliferation of vascular SMCs and signal transduction pathways to which they are coupled.7 However, the transcriptional regulation of phenotypic modulation in vascular SMCs is poorly understood.
Among the most interesting genes shown to be associated with the phenotypic change of vascular SMCs is the smooth muscle myosin heavy chain (MHC) gene. We and others have previously shown that normal medial SMCs in rabbit arterial wall predominantly express either SM1 or SM2, which are generated by an alternative splicing mechanism from a single gene, SM1/2 gene.8 9 10 11 12 13 A recent study has indicated that the SM1/2 gene is expressed exclusively in smooth muscle-containing tissues throughout development.14 SM1 is constitutively expressed at all developmental stages, whereas SM2 appears only after birth. On the other hand, the neointima resulting from vascular injury, including fat feeding or balloon angioplasty, or in vein graft and transplant atherosclerosis, expresses SMemb/NMHC-B, an embryonic isoform of MHC, the expression of which is prominent in fetal aorta and normally downregulated during development.10 Studies on the SMC phenotypes in human coronary arteries demonstrated that expression of SM2 is increased with development but decreased in activated SMCs after angioplasty.15 These studies suggest that the smooth muscle MHC isoforms serve as excellent molecular markers for identifying the diversity of vascular SMC phenotypes in both humans and experimental animals during development and in vascular diseases.
During the last decade, a great deal of progress has been made in
understanding the molecular mechanisms that regulate the striated
muscle lineage-specific gene expression.16 Efforts to
identify the transcription factors that regulate cell type and
lineage-specific gene expression in skeletal muscle have led to the
discovery of master regulatory factors such as the
MyoD/myogenin/myf-5/MRF4 family.17 18 In contrast,
relatively little has been known about the cis-acting
sequences and trans-acting factors that control smooth
muscle-specific gene expression. Recent studies with transient
transfection assays of promoter-reporter constructs into cultured
vascular SMCs have identified the important cis-regulatory
sequences within the 5'-flanking region for directing transcription of
the SM1/2 gene in a smooth muscle-specific manner.19 20 21 22
More recently, promoter regions of other SMC genes, including SM22
and smooth muscle -actin have been characterized by both in vitro
and in vivo experiments.23 24 25 26 27 These studies have
suggested that the CArG box/serum response factor binding sequence is
important for their expression in SMCs.
In contrast to SM1 and SM2 isoforms, expression of 2 nonmuscle MHC isoforms, NMHC-A and SMemb/NMHC-B, is not restricted to SMCs.28 Notably, distribution of each isoform is quite distinct. NMHC-A is expressed in a variety of cell types and is seen as a constitutively expressed "housekeeping" protein required for the structure of the cytoskeleton, whereas expression of the SMemb/NMHC-B gene appears to be more tightly regulated both in terms of cell-type specificity and developmental timing.29 In addition to the downregulation of expression levels in aorta and restricted expression to selected tissues, the induced expression of the SMemb/NMHC-B gene in vascular diseases enables this gene to serve as a model system for identifying the mechanisms of phenotypic change of vascular SMCs. Presumably there exist trans-acting factors that interact with the SMemb/NMHC-B gene and influence its expression in a specific manner.
We have recently characterized the SMemb/NMHC-B promoter and delineated a cis-regulatory element that controls the expression of this promoter in vascular SMCs.30 We mapped an essential sequences for promoter activity to the 15-bp spanning from -105 to -91, designated SE1. In the present study, we have used the oligonucleotide that contains SE1 to screen an expression cDNA library from C2/2 cells,31 an SMC line derived from rabbit aorta, and isolated BTEB2, a zinc finger transcription factor.32 We demonstrate that basic transcriptional element (BTE) binding protein-2 (BTEB2) binds to the SE1 and regulates transcription of the SMemb gene. BTEB2 expression is tissue restricted, with abundant expression in smooth muscle tissues, including intestine, bladder, and uterus, and is scarcely expressed in heart, kidney, spleen, and skeletal muscle. Furthermore, BTEB2 mRNA levels are downregulated in the aorta during development. Immunoreactivity to BTEB2 was clearly increased in the neointimal layer after balloon injury in rat aorta. Thus, BTEB2 defines a transcription factor regulating the SMemb/NMHC-B gene and may play a role in phenotypic modulation of vascular SMCs associated with vascular diseases.
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Materials and Methods |
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Cell Culture
C2/2 cells,31 an established cell line derived from rabbit aortic SMCs, were grown in DMEM (GIBCO-BRL) supplemented with 5% FCS, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cultures were incubated at 37°C in 95% air/5% CO2. NIH3T3, COS-7, and A10 cells were obtained from the American Type Cell Culture Collection and grown in DMEM supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin (GIBCO-BRL).
cDNA Cloning and Sequencing of Rabbit BTEB2
A gt11 cDNA library from C2/2 cell mRNA was screened with
modifications of the methods originally described by Singh et
al.33 Two complementary oligonucleotides,
5'-AATTCATGAGGGC-CAGCCTATGAGATTGGGACTTCGGTGG-CCTG-3'
and 5'-ATTCCCAATCTCATAGGCTGGCCCTCATGCC-3', which contain
the SE1 (indicated by underlining), an essential sequence for the
rabbit SMemb/NMHC-B promoter,30 were annealed,
phosphorylated, and ligated. Ligated products
containing 5 tandem repeats of the oligonucleotide were
isolated by acrylamide gel electrophoresis and cloned into
the EcoRI site of pBluescript II (Stratagene). The DNA
fragment containing 5 tandem repeats of the
oligonucleotide was isolated from this plasmid,
32P labeled, and used as a probe for screening.
Approximately 1 million phage clones were plated and transferred to
nylon membranes. Of 11 positive clones isolated, 3 corresponded to
BTEB2. The other 3 clones were identified as overlapping cDNAs of
another gene. The largest of these 3 was cloned into pBluescript II and
used to generate a probe for screening a rabbit fetal aortic cDNA
library in the EcoRI site of ZAPII (Clontech
Laboratories, Inc) to obtain the full-length clone, termed BTEB2A.
Both strands of the entire rabbit BTEB2 cDNA were sequenced on an
automated DNA sequencer using the dye termination method.
Preparation of Glutathione S-Transferase
(GST)-Fused BTEB2
A bacterial expression plasmid encoding a fusion protein between
GST and amino acids 1 through 219 of the BTEB2 molecule was prepared by
the polymerase chain reaction (PCR). The
oligonucleotide 5'-CCCGAATTCATGCCCAGTTCTACAAACCAG-3'
was used as the forward primer, the oligonucleotide
5'-GGGCTCGAGTCAGTTCTGGTGCCTCTTCAT-3' was used as the reverse primer,
and BTEB2A phagemid was used as the template in the PCR. The PCR
product was digested with EcoRI and XhoI and
cloned inframe into pGEX-4T-1 vector (Pharmacia). The resulting
construct, pGEX/BTEB2, was used to transform Escherichia
coli strain BL21, and GST-BTEB2 synthesis was induced with 1
mmol/L isopropylthiogalactopyranoside (IPTG) for 24 hours at 20°C.
The fusion protein was purified from bacterial extracts by affinity
chromatography on glutathione-agarose.
Plasmid Constructions and Transfections
Plasmids Del-105, Del-99, Del-89, Del-36, pGVm1, pGVm2, pGVm3,
and pGVm4 have been described.30 Eukaryotic
expression vector pcDNA/BTEB2 was constructed by PCR amplification of
BTEB2 cDNA obtained from BTEB2A with the same primers as those used
for pGEX/BTEB2 construction. The expression of BTEB2 was directed by
cytomegalovirus promoter. For transient transfection assays, COS-7
cells were transfected by a modified calcium phosphate-DNA
coprecipitation method as previously described.30 Each
35-mm-diameter culture dish of 1x105 cells was transfected
with 1 µg of a luciferase reporter plasmid and 1 µg of
either vector alone or pcDNA/BTEB2. Transfected cells were harvested
for extract preparation at 48 hours after transfection. Transfections
were performed in duplicate in at least 3 independent experiments. Cell
extracts were prepared by luciferase assay kit (PicaGene system, Nippon
Gene), and levels of luciferase activity were measured with a
luminometer (Lumat LB9501, Berthold). Protein concentration was
measured by using the BCA protein assay reagent (Pierce) with BSA as
standard. The luciferase activity was normalized to protein
concentration of the cell lysates.
Electrophoretic Mobility Shift Assays (EMSAs)
The extraction of nuclear proteins was performed with
modifications of the methods originally described by Dignam et
al.34 Briefly, cells were washed twice in cold PBS and
scraped, and the cellular pellet was resuspended in (in mmol/L)
HEPES (pH 7.6) 10, KCl 10, and MgCl2 1.5 on ice. Cells were
homogenized with 10 strokes of Dounce
homogenizer (type B pestle) and centrifuged to
collect the nuclei, which were subsequently resuspended in 20
mmol/L HEPES (pH 7.6), 0.42 mol/L NaCl, 5 mmol/L
MgCl2, and 25% glycerol to allow elution of nuclear
proteins by gentle shaking on ice for 30 minutes. Nuclear extracts were
pelleted for 15 minutes at 15 000 rpm at 4°C, and supernatant was
then dialyzed against (in mol/L) HEPES (pH 7.6) 20, EDTA 0.2, KCl 50,
and MgCl2 5, and 20% glycerol overnight. Nuclear extracts
were then pelleted for 30 minutes at 15 000 rpm at 4°C, and the
supernatant was then aliquoted in liquid nitrogen and stored at
-80°C until use. All solutions contained the protease
inhibitors leupeptin and aprotinin at 2 µg/mL, PMSF at
1 mmol/L, and DTT at 1 mmol/L. For gel-shift assays, 10-µg
portions of nuclear extracts or 1 µg of GST fusion proteins were
incubated in 25 µL of binding reaction that contained (in
mmol/L) Tris-HCl (pH 7.5) 10, NaCl 50, and DTT 0.5, and 10% glycerol,
0.05% NP-40, and 2 µg of poly(dI-dC) for 20 minutes at room
temperature to titrate out nonspecific binding before the addition of
15 000 to 20 000 cpm of labeled oligonucleotide; the
reaction mixture was then further incubated for 20 minutes. When
unlabeled oligonucleotides or antibodies were added,
nuclear extracts were preincubated for 20 minutes at room temperature
or for 30 minutes at 4°C, respectively, before the addition of
labeled probe. Samples were loaded on a prerun 5%
polyacrylamide gel in 0.5x TBE (45 mmol/L Tris borate and
1 mmol/L EDTA) and electrophoresed at 150 V. The gels were then
dried and exposed to Kodak XAR film at -80°C.
RNA Extraction and Northern Analysis
Total cellular RNA was prepared from organ samples and from
cultures of the rabbit aortic SMC line C2/2, the rat SMC line A10, and
non-SMC lines, including murine NIH3T3 cells and monkey kidney-derived
COS-7 cells, by the single-step guanidinium thiocyanate protocol
described previously.35 Total RNA was fractionated in
1.2% agarose/formaldehyde gels. Northern blotting was performed using
20 µg of RNA/sample as described previously.20 RNA was
transferred to nylon membrane (Hybond N+, Amersham) as
described by the manufacturer. After being UV cross-linked, blots were
stained with 0.04% methylene blue to permit quantification of the 28S
and 18S ribosomal RNA subunits in each lane. Probes included a 664-bp
fragment that contains an entire coding region of the rabbit BTEB2 cDNA
and a 1.2-kb fragment of human Sp-1 cDNA probe. Filter hybridizations
were carried out in a solution of 50% formamide, 5x SSPE, 10x
Denhardt's solution, 1% SDS, and 0.1 mg/mL herring sperm DNA at
42°C. Filters were washed to a final stringency of 0.1x SSC at
42°C and exposed to Kodak XAR film at -80°C for 48 hours. Antibody
against Sp1 (PEP2) was obtained from Santa Cruz Biotechnologies.
BTEB2 Antibody and Western Blot Analysis
A short peptide specifying the carboxyl terminal end of BTEB2
(Asp-His-Leu-Ala-Leu-His-Met-Lys-Arg-His-Gln-Asn) was synthesized and
used for immunization. The synthetic short peptide was conjugated with
BSA and injected subcutaneously into rabbits at biweekly intervals.
Titers of the antisera were determined by an ELISA. Tissues from either
neonatal or adult rats were pulverized under liquid nitrogen. These
tissues and cells were incubated in ice-cold sample buffer containing
2% SDS, 0.1 mol/L DTT, and 60 mmol/L Tris-HCl (pH 6.8) for 30
minutes. Equal amounts (20 µg of total protein) of each lysate were
boiled for 5 minutes, separated on 8% SDS-PAGE, and then transferred
to nitrocellulose (Hybond-ECL, Amersham Corp). The membrane was blocked
for 2 hours at room temperature with 10% nonfat dried milk in
Tris-buffered saline containing 0.1% (wt/vol) Tween 20 (TBS-T). The
blots were incubated for 1 hour at room temperature with anti-BTEB2
antibody and followed by incubation for 1 to 2 hours with horseradish
peroxidase-labeled goat anti-rabbit IgG in TBS-T containing 2% nonfat
dried milk. The immunoreactive bands were visualized by enhanced
chemiluminescence (Amersham Life Science, Inc.).
RNase Protection Assay
The 274-nucleotide EcoRI-SacI
fragment of the rabbit BTEB2 cDNA clone was subcloned into pBluescript
II. After linearizing the plasmid DNA with HindIII, the RNA
probe was synthesized with T3 RNA polymerase and
[-32P]UTP according to the manufacturer's protocols
as described for the Riboprobe Gemini System II kit (Promega). Total
RNA (5 µg) was hybridized with the RNA probe. The RNase protection
assay was carried out according to the manufacturer's protocols as
described for the ribonuclease protection assay kit (Ambion). The probe
and protected fragments were analyzed on a denaturing urea-5%
polyacrylamide gel.
Immunohistochemistry
Balloon injury of the aorta was performed in adult male Wister
rats (300 to 350 g) under general anesthesia. A 2F
balloon embolectomy catheter was introduced via the femoral artery and
advanced to the level of the thoracic aorta. The balloon was then
inflated and the catheter withdrawn along the length of the
thoracoabdominal aorta. The balloon was deflated, and the procedure was
repeated 3 times. Rats were euthanized at 2 weeks after injury. The
aorta was fixed in 10% formalin and paraffin embedded.
Immunohistochemistry using anti-BTEB2 antibody was carried out by using
the Vectastain Elite ABC kit (Vector Laboratories).
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Results |
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Isolation of Rabbit BTEB2 cDNA
We have recently shown that the 15-bp DNA sequence located at -105, to which we refer as SE1 (sequence element-1), is important for a high level of expression of the SMemb/NMHC-B gene.30 To isolate cDNA clones encoding proteins that bind to the SE1 sequence, we constructed an expression cDNA library from C2/2 cells, an SMC line derived from rabbit aorta, in the
gt11 vector. We chose C2/2 cells
because our previous experiments suggested that they contain factors
that can modulate SMemb/NMHC-B promoter activity.20 The
screening was performed with concatenated
oligonucleotides that contain the SE1 sequence. From
~1 million phage plaques, 11 clones that exhibited binding to SE1
were isolated. Sequence analysis revealed that 3 of these
clones contain independent, partial cDNA inserts derived from the BTEB2
mRNA. Sequences encoding the missing coding region of the gene were
obtained by screening a rabbit fetal aortic cDNA library using this
clone as a probe. We finally obtained a 2.3-kb cDNA insert, which
contained the full-length coding region of the rabbit BTEB2 cDNA.
Analysis of the predicted open reading frame encoding 219 amino
acids revealed 3 putative zinc finger domains (Figure 1
). Sequence comparisons showed a high
homology to human BTEB2 with 93.6% and 98.6% sequence identity at the
nucleotide level and amino acid level, respectively.
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BTEB2 Is Present in the Nuclear Factor Complex That Binds to
the SE1
In our previous EMSAs performed with nuclear extracts from C2/2
cells, SE1 sequence has been shown to form a single complex that was
competed by a sequence for CCAAT binding protein but not for Sp1 or
Sp1-related proteins.30 However, C2/2 nuclear extracts
prepared by the modified version of the original procedure described by
Dignam et al34 have yielded 2 shifted complexes (C1 and
C2, Figure 2), both of which comigrated
with the complexes formed by the canonical Sp1 binding site that was
used a reference. The formation of these complexes was sequence
specific, because a 5-bp substitution mutation within SE1 sequence,
from GGGCC to TTTAA located between -105 and -101, lost its ability
to compete for the binding with SE1 sequence (Figure 2A). Competition
with excess unlabeled oligonucleotides containing Sp1
or BTEB sequences indicated that SE1 is homologous to the binding
sequences for Sp1 or BTEB1. Most notably, incubation with the BTEB2
antibody gave rise to a supershifted complex, which indicates that
BTEB2 is a component of the complexes C1 and C2 (Figure 2B). The
specificity of this supershifted complex was supported by the
observations that rabbit preimmune serum has no effects on the factor
bindings with SE1 probe, and BTEB2 antibody has no effects on the
interaction between Sp1 and its consensus binding sequences. These
results unequivocally demonstrate that BTEB2 is present in the
nuclear factor:SE1 complexes. In addition, we found that SE1 can also
serve as a binding site for Sp1, because addition of Sp1 antibody
yielded supershifted bands (Figure 2C).
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DNA Binding Activity of GST-BTEB2 Protein
Further confirmation that BTEB2 is an SE1
sequence-binding protein was obtained by incubating the GST-BTEB2
protein with the labeled oligonucleotide containing SE1
sequence (see Table for sequence). The
oligonucleotide EM0 was retarded by GST-BTEB2 and not
by GST alone in a gel-shift assay (Figure 3A). Binding to EM0 was completely
competed by a 500-fold molar excess of unlabeled EM0. To define the
sequence to which BTEB2 binds, duplex oligonucleotides,
EM1 to EM5, were used as competitors. The complex was efficiently
competed with a 500-fold molar excess of unlabeled EM1, which keeps the
sequence from -105 to -96 unchanged. Unlabeled EM2, which keeps the
sequence from -103 to -94 unchanged, competed for binding but less
efficiently. EM3, EM4, and EM5 with unchanged sequences from -100 to
-91, -115 to -101, and -94 to -85, respectively, did not compete.
These results indicate that a sequence from -105 to -100 is required
for GST-BTEB2 to bind to the EM0. This was further confirmed by testing
the binding of GST-BTEB2 to radiolabeled EM0 to EM5 (Figure 3B). A
DNA:GST-BTEB2 complex was formed with either EM0 or EM1, but not with
EM2, EM3, EM4, or EM5.
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Given that BTEB2 is expected to bind GC-rich sequences,32
we tested whether the GST-BTEB2 protein specifically binds to consensus
sequences of the Sp1-binding site,36
CACC-box,37 or BTEB site,38 each of which is
known to be a binding site for Sp1 or Sp1-related zinc finger
transcription factors. Indeed, the shifted complex formed with
EM0 and GST-BTEB2 protein was completely abolished by addition
of unlabeled oligonucleotide carrying an Sp1
binding site, CACC box, or BTE binding site, but not by the unlabeled
oligonucleotide containing the binding sites for CAT
(Figure 3C
).
Transactivation of the SMemb/NMHC-B Promoter by BTEB2
Given the capability of BTEB2 to bind to the SE1, an element
important for the expression of the SMemb/NMHC-B gene, we determined
whether BTEB2 activates transcription from the SMemb/NMHC-B
promoter. The full-length BTEB2 cDNA under the control of the
cytomegalovirus promoter was cotransfected into COS-7 cells with a
series of 5'-deletion constructs of rabbit SMemb/NMHC-B promoter
(Figure 4). Overexpression of BTEB2
resulted in a significant induction of Del-105 reporter gene expression
(7.5±0.4–fold). Deletion of a sequence to -99 bp resulted in a
marked decrease in transactivation by BTEB2 (2.1±0.2–fold). To
address further the effects of BTEB2 on the SMemb/NMHC-B promoter
activity, mutations were introduced into SE1 sequence within the
context of Del-138, which contains SMemb/NMHC-B promoter sequence up to
-138 bp. Luciferase activity of Del-138 reporter gene was
significantly increased by BTEB2 (7.4±0.4-fold) (Figure 5). Mutations of sequence either between
-105 and -101 (pGVm1) or between -100 and -96 (pGVm2) resulted in a
noticeable reduction of fold activation by BTEB2. In contrast,
mutations of sequence either between -95 and -91 (pGVm3) or between
-90 and -86 (pGVm4) failed to block the BTEB2-induced expression of
the reporter gene. Overall, these results indicate that BTEB2 can
activate SMemb/NMHC-B promoter activity and the sequence
between -105 and -96 mediates at least in part the induction by
BTEB2.
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To verify that SE1 sequence mediates the activation of the SMemb promoter by BTEB2, we inserted 5 copies of SE1 sequence upstream of the simian virus 40 promoter-luciferase vector pGVC to generate the construct 5xSE-pGVC. Cotransfection of BTEB2 expression vector stimulated transcription of 5xSE-pGVC by 6.2-fold, whereas it activated pGVC by 2.8-fold (data not shown). These results indicate that SE1 sequence confers the BTEB2-induced activation on the heterologous promoter.
Regulation of SMemb Promoter Activity by Phorbol
12-Myristate 13-Acetate (PMA)
Having demonstrated that overexpression of BTEB2 leads to the SE1
sequence-dependent activation of the SMemb promoter, we examined
whether this promoter is also responsive to stimuli that induce
endogenous BTEB2 expression. Stimulation of C2/2 cells with
PMA leads to the sustained increase in BTEB2 mRNA levels (K.-K. Kowase,
M. Kurabayashi, R. Nagai, unpublished results, 1999). Figure 5C
shows that PMA increases luciferase activity derived form wild-type
SMemb promoter, and this effect of PMA was attenuated when the 10-bp
sequence located between -105 and -96 was mutated (see pGVm1, pGVm2
in Figure 5C).
Expression of the BTEB2 in Adult Tissues and Developing
Aorta
To determine the expression profile of BTEB2 mRNA, we performed
Northern blot analysis of RNAs from various tissues of adult
rabbit. The mRNA is expressed most abundantly in the small and large
intestines, followed by lung, bladder, and uterus, but not in cerebrum,
ventricle, liver, skeletal muscle, aorta, spleen, and kidney (Figure 6A). A more refined analysis of
BTEB2 mRNA distribution was achieved by RNase protection assay (Figure 6B). A fully protected band was clearly detected only in the samples
from small intestine and lung, whereas a faint band was present in
the liver, and no signal was detected in the cerebrum, atrium,
ventricle, spleen, skeletal muscle, and kidney. We next examined the
expression of BTEB2 mRNA in various cell lines, such as C2/2, NIH3T3,
COS-7, and A10 cells. BTEB2 was abundantly expressed in C2/2 cells and
scarcely expressed in other cells (Figure 6C). Furthermore, the RNase
protection assay revealed that BTEB2 mRNA levels are downregulated in
developing rabbit aorta; BTEB2 mRNA is abundantly expressed in fetal
aorta but barely detectable in adult aortas (Figure 6D).
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BTEB2 Protein Expression in a Developing Aorta and an Injured
Aorta
Presence of BTEB2 protein was determined by immunoblot
analysis (Figure 7). The
specificity of the BTEB2 antibody was confirmed by its ability to
recognize GST-BTEB2 fusion protein but not GST (Figure 7A). The BTEB2
antibody detected a major band, the molecular mass of which was
35 kDa in C2/2 cells (Figure 7B), although the calculated molecular
mass of BTEB2 protein deduced from its cDNA sequence is 23 kDa.
BTEB2 could be easily detected in neonatal aorta, but not in adult
aorta. From these results, we conclude that BTEB2 is expressed in a
tissue-restricted and developmentally regulated manner in the aorta. To
examine the induction of BTEB2 in injured vessels, balloon injury of
rat aortas was performed as described (see Materials and Methods). At 2
weeks after balloon injury, SMCs in the neointima were
positive for BTEB2 (Figure 7C) as well as for SM1 and SMemb (data not
shown). In the medial SMCs, however, only a few cells adjacent to the
internal elastic lamina were positive for BTEB2.
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Discussion |
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We undertook this study as a first approach toward identifying transcription factors that contribute to phenotypic modulation of vascular SMCs. We have recently shown that the 15-bp sequence located between -105 and -91, designated SE1, is important for directing transcription of the SMemb/NMHC-B gene in vascular SMCs but not in other cell types, including NIH3T3 and COS-7 cells.30 In the present study, using the concatenated SE1 sequence as a probe, we have screened an expression cDNA library prepared from an SMC line. We have isolated the cDNA clones encoding BTEB2, a C2H2 zinc finger transcription factor that has previously been cloned from human placenta, and explored the role for BTEB2 in regulating SMemb/NMHC-B gene expression.32 Our studies have defined BTEB2 as a transcription factor that regulates the SMemb/NMHC-B gene on the basis of the following 4 different criteria. (1) We verified that BTEB2 is a component of nuclear factor:DNA complexes formed with SE1 probe by the supershift assays using the BTEB2 antibody as well as by the gel-shift assays of bacterially expressed GST-BTEB2 fusion proteins. Consistent with the previous report in which BTEB2-binding sequence is similar or identical to Sp1-binding sequence,38 binding of BTEB2 to SE1 sequence was efficiently competed by excess amounts of unlabeled Sp1 or Sp1-related factor-binding sequences, such as CACC box and BTE. (2) Cotransfection analysis showed that BTEB2 significantly activates the SMemb/NMHC-B promoter/luciferase reporter gene. (3) BTEB2 expression is tissue restricted in adult rabbit and developmentally downregulated in the aorta as assessed by Northern blot and RNase protection analyses. (4) BTEB2 protein is reinduced in the neointimal layer after balloon injury in adult aorta. This pattern of expression correlates remarkably with that of SMemb/NMHC-B.9 These lines of evidence suggest that BTEB2 participates in the regulation of SMemb/NMHC-B gene expression and may play an important role in modulating the phenotypes of vascular SMCs.
It should be noted that we originally characterized the binding protein to SE1 to be a member of CCAAT binding.30 However, by modifying the protocol of nuclear extract preparation, we found that besides CCAAT binding protein, Sp1-like proteins could also bind to this element, which turned out to be BTEB2 in the present study.
BTEB2 belongs to the Krüppel family of transcription factors, which contain 3 C2H2 zinc finger domains. The family of C2H2 zinc finger genes represent a class of DNA binding proteins, many of which have been demonstrated to have roles in regulating transcription in diverse genes.39 40 BTEB2 has been implicated in the regulation of transcription of eukaryotic genes on the basis of cotransfection analysis using BTEB2 expression vector and SV40 early promoter construct which contains GC-rich sequence.32 However, cellular genes specifically regulated by BTEB2 and the function of BTEB2 in physiologically relevant context have remained to be determined. In the present study, we found that tissue distribution of BTEB2 is clearly different from that of Sp1 in that BTEB2 is abundantly expressed in smooth muscle tissues, whereas Sp-1 is almost ubiquitously expressed.41 Because there exist many transcription factors that recognize the GC-rich sequence as a binding site, it is difficult to ascribe a specific function to particular members of GC-box binding proteins. In the case of BTEB2, its unique features in tissue distribution and developmental regulation allow us to speculate that BTEB2 plays a role in regulating the SMC genes the expression of which is developmentally regulated. Conclusively establishing a specific role of BTEB2 will need to wait for studies on the consequences of the targeted disruption of this gene.
Recently, several C2H2 zinc finger genes that are implicated in the regulation of tissue-specific gene expression have been cloned. The cDNA encoding a Krüppel-related polypeptide, designated EKLF, was shown by both a biochemical and a genetic approach to represent a CACC binding protein that controls the ß-globin gene.42 43 The BKLF/TEF-2, another member of Krüppel family of transcription factors, which binds strongly to CACC box, contains a unique basic region and its expression is less tissue restricted.44 More recently, 2 other genes that contain structural homology to BTEB2 have been reported, LKLF and GKLF, which are preferentially expressed in lung and gut, respectively.42 45 The amino acid sequence of the zinc finger domain is remarkably conserved among the Krüppel family of transcription factors. Although any specific function of each of these factors remains to be determined, we observed that both LKLF and GKLF are expressed in the aorta and are able to activate either SMemb/NMHC-B or SM1/2 promoters in transient transfection assays (data not shown). These observations support the notion that certain members of Krüppel-like zinc finger proteins, including BTEB2, LKLF, and GLKF, can potentially play a physiologically significant role in the control of SMC genes.
We also found that BTEB2 expression in the balloon-injured aorta is confined to the neointima, where SMCs exhibit the activated or synthetic phenotype. Recently, the transcription factors that display SMC phenotype-restricted expression have been cloned. HoxB7 and HoxC9 show preferential mRNA expression in fetal SMCs.46 MEF2A, MEF2B, and MEF2D expression in smooth muscle is associated with phenotypically activated SMCs.47 Interestingly, like BTEB2, MEF2 expression is upregulated in neointima in a pattern similar to that of BTEB2 after balloon injury. It is tempting to speculate that induced expression of BTEB2, in conjunction with that of MEF2s and other injury-induced transcription factors, such as Ets-148 and Egr-1, may play a role in regulating the activated SMC phenotype-specific pattern of gene expression in neointima. Indeed, we have recently found that BTEB2 is capable of activating transcription of the c-jun and Id2 genes, which are implicated in cellular proliferation (M. Kurabayashi et al, unpublished data, 1999). Thus, it is possible that BTEB2 mediates the proliferative response after balloon injury.
In conclusion, we have identified BTEB2 as a transcription factor regulating SMemb/NMHC-B gene expression. Because BTEB2 expression is associated with the activated phenotype of vascular SMCs, our present data should provide the insight into the molecular mechanisms of phenotypic modulation underlying the development of vascular diseases.
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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. Kurabayashi and R.N.). We thank Dr H. Katoh for preparation of the BTEB2 antibody. We also thank Miki Yamazaki and Kaori Ishihara for their excellent technical help.
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Footnotes |
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This manuscript was sent to Laurence H. Kedes, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
© 1999 American Heart Association, Inc.
Received December 29, 1997; accepted April 22, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Dzau VJ, Gibbons GH, Cooke JP, Omoigui N. Vascular biology and medicine in the 1990s: scope, concepts, potentials, and perspectives. Circulation. 1993;87:705–719.
2.
Owens GK. Regulation of differentiation of vascular
smooth muscle cells. Physiol Rev. 1995;75:487–517.
3. Frid MG, Shekhonin BV, Koteliansky VE, Glukhova MA. Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin. Dev Biol. 1992;153:185–193.[Medline] [Order article via Infotrieve]
4.
Glukhova MA, Frid MG, Koteliansky VE. Developmental
changes in expression of contractile and cytoskeletal proteins in human
aortic smooth muscle. J Biol Chem. 1990;265:13042–13046.
5. Glukhova MA, Frid MG, Koteliansky VE. Phenotypic changes of human aortic smooth muscle cells during development and in the adult vessel. Am J Physiol. 1991;261(suppl 4):78–80.
6.
Ueki N, Sobue K, Kanda K, Hada T, Higashino K.
Expression of high and low molecular weight caldesmons during
phenotypic modulation of smooth muscle cells. Proc Natl Acad Sci
U S A. 1987;84:9049–9053.
7. Ross R. Growth regulatory mechanisms and formation of the lesions of atherosclerosis. Ann N Y Acad Sci. 1995;748:1–4.
8.
Nagai R, Kuro-o M, Babij P, Periasamy M.
Identification of two types of smooth muscle myosin heavy chain
isoforms by cDNA cloning and immunoblot analysis.
J Biol Chem. 1989;264:9734–9737.
9.
Kuro-o M, Nagai R, Tsuchimochi H, Katoh H, Yazaki Y,
Ohkubo A, Takaku F. Developmentally regulated expression of vascular
smooth muscle myosin heavy chain isoforms. J Biol Chem. 1989;264:18272–18275.
10.
Kuro-o M, Nagai R, Nakahara K, Katoh H, Tsai RC,
Tsuchimochi H, Yazaki Y, Ohkubo A, Takaku F. cDNA cloning of a myosin
heavy chain isoform in embryonic smooth muscle and its expression
during vascular development and in
arteriosclerosis. J Biol Chem. 1991;266:3768–3773.
11.
Babij P, Kelly C, Periasamy M. Characterization of a
mammalian smooth muscle myosin heavy-chain gene: complete
nucleotide and protein coding sequence and analysis
of the 5' end of the gene. Proc Natl Acad Sci U S A. 1991;88:10676–10680.
12. Babij P, Periasamy M. Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing. J Mol Biol. 1989;210:673–679.[Medline] [Order article via Infotrieve]
13.
Simons M, Wang M, McBride OW, Kawamoto S, Yamakawa K,
Gdula D, Adelstein RS, Weir L. Human nonmuscle myosin heavy chains are
encoded by two genes located on different chromosomes. Circ
Res. 1991;69:530–539.
14.
Miano JM, Cserjesi P, Ligon KL, Periasamy M, Olson EN.
Smooth muscle myosin heavy chain exclusively marks the smooth muscle
lineage during mouse embryogenesis. Circ Res. 1994;75:803–812.
15. Aikawa M, Kim HS, Kuro-o M, Manabe I, Watanabe M, Yamaguchi H, Yazaki Y, Nagai R. Phenotypic modulation of smooth muscle cells during progression of human atherosclerosis as determined by altered expression of myosin heavy chain isoforms. Ann N Y Acad Sci. 1995;748:578–585.[Medline] [Order article via Infotrieve]
16.
Olson EN. Regulation of muscle transcription by the
MyoD family: the heart of the matter. Circ Res. 1993;72:1–6.
17.
Weintraub H, Davis R, Tapscott S, Thayer M, Krause R,
Benezra R, Blackwell TK, Turner D, Rupp R, Hollenberg S, Zhuang YAL.
The MyoD gene family: nodal point during specification of the muscle
cell lineage. Science. 1991;251:761–766.
18. Weintraub H, Davis R, Tapscott S, Thayer M, Krause M, Benezra R, Blackwell TK, Turner D, Rupp R, Hollenberg S, Zhuang Y, Lassar A. The MyoD gene family: nodal point during specification of the muscle cell lineage. Science. 1991;251:761–766.
19.
Katoh Y, Loukianov E, Kopras E, Zilberman A, Periasamy
M. Identification of functional promoter elements in the rabbit smooth
muscle myosin heavy chain gene. J Biol Chem. 1994;269:30538–30545.
20.
Watanabe M, Sakomura Y, Kurabayashi M, Manabe I, Aikawa
M, Kuro-o M, Suzuki T, Yazaki Y, Nagai R. Structure and
characterization of the 5'-flanking region of the mouse smooth muscle
myosin heavy chain (SM1/2) gene. Circ Res. 1996;78:978–989.
21.
White SL, Low RB. Identification of promoter elements
involved in cell-specific regulation of rat smooth muscle myosin heavy
chain gene transcription. J Biol Chem. 1996;271:15008–15017.
22.
Madsen CS, Hershey JC, Hautmann MB, White SL, Owens GK.
Expression of the smooth muscle myosin heavy chain gene is regulated by
a negative-acting GC-rich element located between two positive-acting
serum response factor-binding elements. J Biol Chem. 1997;272:6332–6340.
23.
Solway J, Seltzer J, Samaha FF, Kim S, Alger LE, Niu Q,
Morrisey EE, Ip HS, Parmacek MS. Structure and expression of a smooth
muscle cell-specific gene, SM22. J Biol Chem. 1995;270:13460–13469.
24.
Shimizu RT, Blank RS, Jervis R, Lawrenz SS, Owens
GK. The smooth muscle -actin gene promoter is differentially
regulated in smooth muscle versus non-smooth muscle cells. J
Biol Chem. 1995;270:7631–7643.
25. Kim S, Ip HS, Lu MM, Clendenin C, Parmacek MS. A serum response factor-dependent transcriptional regulatory program identifies distinct smooth muscle cell sublineages. Mol Cell Biol. 1997;17:2266–2278.[Abstract]
26.
Joseph LL, Miano M, Mercer B, Olson EN. Expression of
the SM22 promoter in transgenic mice provides evidence for distinct
transcriptional regulatory programs in vascular and visceral muscle
cells. J Cell Biol. 1996;132:849–859.
27.
Joseph LL, Miano M, Cserjesi P, Olson EN. SM22a, a
marker of adult smooth muscle, is expressed in multiple myogenic
lineages during embryogenesis. Circ Res. 1996;78:188–195.
28.
Kawamoto S. Neuron-specific alternative splicing of
nonmuscle myosin II heavy chain-B pre-mRNA requires a cis-acting intron
sequence. J Biol Chem. 1996;271:17613–17616.
29.
Kawamoto S, Adelstein RS. Chicken nonmuscle myosin
heavy chains: differential expression of two mRNAs and evidence for two
different polypeptides. J Cell Biol. 1991;112:915–924.
30. Manabe I, Kurabayashi M, Simomura Y, Kuro-o M, Watanabe N, Watanabe M, Aikawa M, Suzuki T, Yazaki Y, Nagai R. Isolation of the embryonic form of smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and characterization of its 5'-flanking region. Biochem Biophys Res Commun. 1997;239:598–605.[Medline] [Order article via Infotrieve]
31.
Sasaki Y, Uchida T, Sasaki Y. A variant derived from
rabbit aortic smooth muscle: phenotype modulation and
restoration of smooth muscle characteristics in cells in culture.
J Biochem. 1989;106:1009–1018.
32.
Sogawa K, Imataka H, Yamasaki Y, Kusume H, Abe H, Fujii
KY. cDNA cloning and transcriptional properties of a novel GC
box-binding protein, BTEB2. Nucleic Acids Res. 1993;21:1527–1532.
33. Singh H, LeBowitz JH, Baldwin AJ, Sharp PA. Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell. 1988;52:415–423.[Medline] [Order article via Infotrieve]
34.
Dignam JD, Lebovitz RM, Roeder RG. Accurate transon
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res. 1988;11:1475–1489.
35. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
36. Kadonaga JT, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987;51:1079–1090.[Medline] [Order article via Infotrieve]
37.
Miller IJ, Bieker JJ. A novel, erythroid cell-specific
murine transcription factor that binds to the CACCC element and is
related to the Krüppel family of nuclear proteins. Mol Cell
Biol. 1993;13:2776–2786.
38.
Sogawa K, Kikuchi Y, Imataka H, Fujii KY. Comparison of
DNA-binding properties between BTEB and Sp1. J Biochem
Tokyo. 1993;114:605–609.
39. Nardelli J, Gibson TJ, Vesque C, Charnay P. Base sequence discrimination by zinc-finger DNA-binding domains. Nature. 1991;349:175–178.[Medline] [Order article via Infotrieve]
40. Pabo CO, Sauer RT. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095.[Medline] [Order article via Infotrieve]
41.
Saffer JD, Jackson SP, Annarella MB. Developmental
expression of Sp1 in the mouse. Mol Cell Biol. 1991;11:2189–2199.
42. Anderson KP, Kern CB, Crable SC, Lingrel JB. Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family. Mol Cell Biol. 1995;15:5957–5965.[Abstract]
43. Bieker JJ, Southwood CM. The erythroid Krüppel-like factor transactivation domain is a critical component for cell-specific inducibility of a ß-globin promoter. Mol Cell Biol. 1995;15:852–860.[Abstract]
44. Crossley M, Whitelaw E, Perkins A, Williams G, Fujiwara Y, Orkin SH. Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells. Mol Cell Biol. 1996;16:1695–1705.[Abstract]
45.
Shields JM, Christy RJ, Yang VW. Identification and
characterization of a gene encoding a gut-enriched Krüppel-like
factor expressed during growth arrest. J Biol Chem. 1996;271:20009–20017.
46.
Miano JM, Firulli AB, Olson EN, Hara P, Giachelli CM,
Schwartz SM. Restricted expression of homeobox genes distinguishes
fetal from adult human smooth muscle cells. Proc Natl Acad Sci
U S A. 1996;93:900–905.
47.
Firulli AB, Miano JM, Bi W, Johnson AD, Casscells W,
Olson EN, Schwarz JJ. Myocyte enhancer binding factor-2 expression and
activity in vascular smooth muscle cells: association with the
activated phenotype. Circ Res. 1996;78:196–204.
48.
Hultgardh NA, Cercek B, Wang JW, Naito S, Lovdahl C,
Sharifi B, Forrester JS, Fagin JA. Regulated expression of the ets-1
transcription factor in vascular smooth muscle cells in vivo and in
vitro. Circ Res. 1996;78:589–595.
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K. Kawai-Kowase, M. Kurabayashi, Y. Hoshino, Y. Ohyama, and R. Nagai Transcriptional Activation of the Zinc Finger Transcription Factor BTEB2 Gene by Egr-1 Through Mitogen-Activated Protein Kinase Pathways in Vascular Smooth Muscle Cells Circ. Res., October 29, 1999; 85(9): 787 - 795. [Abstract] [Full Text] [PDF]
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R. Sun, X. Chen, and V. W. Yang Intestinal-enriched Kruppel-like Factor (Kruppel-like Factor 5) Is a Positive Regulator of Cellular Proliferation J. Biol. Chem., March 2, 2001; 276(10): 6897 - 6900. [Abstract] [Full Text] [PDF]
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P. J. Adam, C. P. Regan, M. B. Hautmann, and G. K. Owens Positive- and Negative-acting Kruppel-like Transcription Factors Bind a Transforming Growth Factor beta Control Element Required for Expression of the Smooth Muscle Cell Differentiation Marker SM22alpha in Vivo J. Biol. Chem., November 22, 2000; 275(48): 37798 - 37806. [Abstract] [Full Text] [PDF]
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T. T. Chou, J. Q. Trojanowski, and V. M.-Y. Lee p38 Mitogen-activated Protein Kinase-independent Induction of gadd45 Expression in Nerve Growth Factor-induced Apoptosis in Medulloblastomas J. Biol. Chem., October 26, 2001; 276(44): 41120 - 41127. [Abstract] [Full Text] [PDF]
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J. J. Bieker Kruppel-like Factors: Three Fingers in Many Pies J. Biol. Chem., September 7, 2001; 276(37): 34355 - 34358. [Full Text] [PDF]
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Y. Oyama, N. Akuzawa, R. Nagai, and M. Kurabayashi PPAR{gamma} Ligand Inhibits Osteopontin Gene Expression Through Interference With Binding of Nuclear Factors to A/T-Rich Sequence in THP-1 Cells Circ. Res., February 22, 2002; 90(3): 348 - 355. [Abstract] [Full Text] [PDF]
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I. Manabe and G. K. Owens Recruitment of Serum Response Factor and Hyperacetylation of Histones at Smooth Muscle-Specific Regulatory Regions During Differentiation of a Novel P19-Derived In Vitro Smooth Muscle Differentiation System Circ. Res., June 8, 2001; 88(11): 1127 - 1134. [Abstract] [Full Text] [PDF]
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