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INTRODUCTION |
Vascular smooth muscle cells
(VSMCs)1 in the adult
arterial media exhibit a differentiated phenotype characterized by
expression of smooth muscle (SM) contractile genes such as SM22
,
SM-myosin heavy chain (SM-MHC),
-SM actin, and calponin (1, 2, 4). In contrast, intimal VSMCs, commonly associated with diseased vascular
tissue in adults, express low levels of these genes and resemble less
differentiated fetal or neonatal VSMCs (1-4). Although genes
characteristic of the differentiated VSMC phenotype have been
identified, the morphogenic factors that regulate VSMC phenotype remain
largely unelucidated (5). Identification of these regulators may shed
light on the mechanisms of VSMC phenotype modulation in the etiology of
vascular diseases.
Transforming growth factor
1 (TGF-
1) exerts multiple effects on
VSMCs in vitro, including a
concentration-dependent effect on proliferation and
stimulation of extracellular matrix protein synthesis (1, 6-8).
Additionally, TGF-
1 potently up-regulates expression of
SM-contractile marker genes in cultured VSMCs, pluripotent C3H10T1/2, and neural crest stem cells
(9-11). Exploration of the molecular mechanism whereby TGF-
1
up-regulates
-SM actin gene expression in vitro led to
the identification of a TGF-
control element (TCE), which is present
in multiple SM-marker gene promoters and shares sequence similarity
with the Sp1 recognition site (9). The 10-bp TCE element is required
for
-SM actin promoter activity in vitro and SM22
promoter activity in vivo (9, 12). The effects of TGF-
1
on VSMC phenotype have been widely investigated; however, few studies
have focused on the effects of other TGF-
-superfamily members, in
particular the bone morphogenetic proteins (BMPs) in the regulation of
VSMC phenotype.
BMPs were initially isolated based on their ability to induce ectopic
bone and cartilage formation in vivo in muscle tissue or
subcutaneous sites of rodents (13). However, BMP expression studies as
well as the analysis of BMP mouse knockout models suggest that BMPs
exert a broad range of biological activities from cell proliferation,
differentiation, and apoptosis to roles during development and that
functional differences exist between different BMPs (14-16). BMP
signaling is mediated via heterochimeric complex formation with type I
and type II serine/threonine kinase receptors, which transduce their
signals via SMAD proteins (17-19). Evidence suggesting a role for BMPs
in VSMC biology is growing. Arterial expression of BMP2 at day 9.5 post-coitus and BMP4 and -6 at day 10.5 post-coitus in mice (20, 21)
correspond with the temporal expression of the SM differentiation
marker genes, h1 calponin, SM22
, and SM-MHC
(22-24). Furthermore, BMP6 expression has been documented in both
human intimal and medial VSMCs (25). Functionally, BMP7 has been
implicated in maintaining VSMC phenotype in occluded rat renal
arteries, whereas BMP2 instructs 10% of neural crest stem cells to
differentiate into SMCs (11, 26). Expression of the type I
TGF-
-superfamily receptors ALK2, -3, -5, and -6 has been shown in
cultured adult rat VSMCs (27). ALK3 and ALK6 serve as BMP receptors,
ALK5 functions as a TGF-
receptor, and ALK2 can be bound by activin
and BMP7 (28, 29). Importantly, mutations in ALK1 result in human type
II hereditary hemorrhagic telangiectasia, a condition characterized by
multiple vascular defects thought to result from a deficiency in VMSC
differentiation and recruitment, whereas mutations in the BMP type II
receptor cause pulmonary hypertension (30, 31). Downstream from the receptors, BMP signaling molecules, SMADs, have been shown to be
essential for vascular development. SMAD5 null mice lack an organized
vasculature and present with large blood vessels containing a paucity
of VSMCs, whereas SMAD6 knockout mice exhibit aortic ossification and
increased blood pressure (32-34). In total these studies strongly
suggest a role for the BMPs in the regulation of VSMC phenotype and/or
vascular development.
Because they differentiate/mature, VSMCs progress through a phenotypic
continuum that can be divided into fetal, neonatal, and adult stages.
During fetal development, cells commit to the VSMC lineage at days
9-10 post-coitus, initiate expression of SM-marker genes, and
proliferate rapidly (5, 22, 23, 24). Neonatal VSMCs exhibit a reduced
proliferative index, increased SM-marker expression, and increased
matrix synthesis, whereas adult VSMCs maintain basal expression levels
of the SM-markers (5). Understanding how VSMC phenotypic modulation is
regulated during development is particularly important, because intimal VSMCs associated with diseased vascular tissue express a phenotype that
resembles that of normal medial VSMCs during fetal and neonatal development (5).
In this study we demonstrate that multiple TGF-
-superfamily members
and their receptors are dynamically co-expressed during VSMC
differentiation and can reciprocally regulate SM-differentiation marker expression via a TCE- dependent mechanism. In
addition, the activity of TGF-
1 can be modulated by other members of
the TGF-
superfamily. Mechanistically, TGF-
-superfamily members, whether positive or negative regulators of SM-marker gene expression, induce binding of the transcription factor KLF4/GKLF to the TCE element, demonstrating the importance of multiple TGF-
-superfamily members in VSMC phenotypic regulation.
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EXPERIMENTAL PROCEDURES |
Morphogens--
Materials were obtained from common commercial
suppliers unless otherwise stated. Human recombinant BMP-2, -4, and -6 were kindly provided by the Genetics Institute (Cambridge, MA). Human recombinant TGF-
1-3 were purchased from Austral Biologicals (San Diego, CA).
Cell Isolation and Culture--
Aortas were obtained from
Wistar-Kyoto rats. RNA for developmental expression profiles was
isolated directly from fresh rat aortas stripped of adventitia and
endothelium at fetal day 17, fetal day 19, neonate day 1, week 2, week
4, week 6, week 8, and adult (week 12).
For tissue culture, the tunica media and endothelium were removed from
adult rat aortas by partial collagenase digestion, and the medial VSMCs
were dispersed enzymatically with collagenase/elastase (4). Dispersed
cells were cultured in M199, supplemented with 10% fetal calf serum,
100 µg/ml streptomycin, 100 units/ml penicillin, and 250 ng/ml
amphotericin B at 37 °C under 5% CO2 atmosphere. Confluent adult rat VSMCs were growth-arrested by serum starvation for
72 h before treatment with TGF-
-superfamily members for 24 or
48 h. Cells were treated with TGF-
1-3 (2.5 ng/ml), BMP2, -4, or -6 (50 ng/ml), or vehicle. TGF-
s were diluted in 5 mM
HCl (vehicle), and BMPs were diluted in 0.1% bovine serum albumin in
phosphate-buffered saline (vehicle).
RNA Extraction and DNase Treatment--
Total cytoplasmic RNA
was isolated directly from aortic or from cultured VSMCs by Nonidet
P-40 lysis. After incubation in lysis buffer (150 mM NaCl,
10 mM Tris (pH 7.4), 1 mM MgCl2,
0.5% NonidetP-40) for 3 min and centrifugation to pellet the nuclei, the supernatant was supplemented with 10% SDS to a final concentration of 1.5% and extracted twice with Tris-equilibrated phenol, and the RNA
was precipitated. Contaminating DNA was removed by incubation with 5 units of DNase I for 1 h at 37 °C.
Reverse Transcriptase (RT)-PCR--
To generate cDNA, total
RNA (5 µg) was reverse-transcribed in a reaction mixture containing
100 ng of oligo(dT)12 primer, 40 units of RT, 20 units of
RNase inhibitor, and 0.1 mM each dNTP (ATP, CTP, GTP, TTP)
in 1× reaction buffer (50 mM Tris (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol) at 42 °C for 1 h.
The reaction profile for PCR was 94 °C for 30s, annealing
(Table I) for 1 min, and 72 °C for 1 min 30 s.
Primer sequences and number of cycles per
primer pair are given in Table I. Gene-specific primers that spanned an
intron were used in amplification reactions (Table I). PCR reaction
sets using GAPDH-specific primers were carried out concurrently for
normalization. PCR profiles for each primer pair were initially
standardized over a series of cycle numbers to ensure that all
experimental reactions were performed within the linear range. All
RT-PCR reactions were performed in duplicate or triplicate. The
identity of PCR products was confirmed by sequencing.
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Table I
PCR primers and conditions
Shown are sequences of primers, annealing temperature used in
amplification reaction, size of PCR product in bp, and number of PCR
cycles utilized. All PCR primers spanned an intron so that the size of
the amplicon indicated that only cDNA was amplified. N = 25%
of each base, I = inosine. N/A, not applicable.
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Quantification of relative expression levels of each product was
carried out using Southern blotting and hybridization with 32P-labeled probes. To generate probes for Southern
hybridization PCR products were cloned into the TA-vector (Invitrogen)
and sequenced to confirm identity. Levels of expression were determined
on a Packard Instant Imager, which determines real time cpm of
designated areas. All experiments analyzed in this manner were
normalized to GAPDH.
Northern Blotting--
RNA (10 µg) was electrophoresed on
agarose gels (1.5%) containing 2.2 mM formaldehyde and
ethidium bromide in buffer containing 20 mM MOPS, 1 mM EDTA, 5 mM sodium acetate, and transferred
to a Hybond NX nylon membrane by capillary transfer.
-SM actin
(3RD2), SM22
(3RF10), and 18 S probes were labeled using an Amersham Biosciences oligo-labeling kit (4). Hybridization was carried out at 65 °C followed by washing in 0.1× SSC, 0.1% SDS at
65 °C, and quantification was performed on a Packard Instant Imager
and normalized to 18 S.
Western Blotting--
VSMCs were solubilized in 100 µl of
0.125 M Tris-HCl (pH 6.8), 4% (w/v) SDS, 10% (v/v)
-mercaptoethanol, 20% (v/v) glycerol, 0.0004% (w/v) bromphenol
blue and incubated at 100 °C for 5 min. The lysates were then passed
through a 25-gauge needle 3 times. Total protein in each lysate was
quantified using the Bio-Rad protein assay system according to the
manufacturer's instructions. Equal amounts of protein were
electrophoresed on a 10% SDS-polyacrylamide gel and then transferred
to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford,
MA) using a Transblot-SD semi-dry transfer cell. Membranes were
incubated with antibodies to
-SM actin (Sigma clone 1A4, 1:1000
dilution), calponin (Sigma clone hCP, 1:1000 dilution) or GKLF (Santa
Cruz, T16x, 1:1000 dilution). Bound antibody was visualized by
incubation with a secondary antibody (Sigma, 1:5000 dilution) coupled
to horseradish peroxidase followed by ECL. To confirm equal protein
loading, duplicate 10% SDS-polyacrylamide gels were silver- or
Coomassie Blue-stained. GKLF Western blots were carried as described
above, except nuclear protein extracts (prepared as described in
McCaffery and Jackson (35)) were resolved on gels.
Transient Transfections--
The pCB80 (p80) rat SM22
and
pCB55 (p55) promoter fragments (36) were cloned into PGL-3 luciferase
reporter vector (Promega). The TCE element was mutated using the
Stratagene QuikChange site-directed mutagenesis kit. Constructs were
confirmed by sequencing and DNA for transfections was prepared using
Endofree DNA preparation kits (Qiagen).
Rat VSMCs were seeded at a density of 6.6 × 104
cells/60-mm plate and 20 h later transfected with 4 µg of DNA
mixed with 20 µl (3 mg/ml) Superfect Reagent (Qiagen) for 2 h.
The media was then changed, and cells were treated with TGF-
1 (2.5 ng/ml), BMP-2, -4, and -6 (50 ng/ml), or vehicle (phosphate-buffered
saline, 0.1% bovine serum albumin) for 48 h. Forty-eight hours
post-transfection, cells were harvested in lysis buffer (Roche
Molecular Biochemicals). Relative luciferase activity was determined on
a luminometer and normalized to total protein levels. Four independent
experiments were carried out. Experiments were normalized to total
protein as because we and others have observed that viral promoters
interfere with SM differentiation-marker promoter responsiveness due to interactions with CArG boxes present in these promoters (37). Data were
analyzed using Student's t test, and differences were considered significant for p < 0.05.
Electrophoretic Mobility Shift Assay (EMSA)--
Growth arrested
VSMCs were treated with BMPs (50 ng/ml), TGF-
1 (2.5 ng/ml) or
vehicle for 4 h, and nuclear extracts were prepared as in
McCaffery and Jackson (35). A double-stranded DNA probe (50 ng) was
labeled with [
-32P]CTP (50 µCi) using Klenow, and
unincorporated nucleotides were removed from the probe using a
nucleotide removal kit (Qiagen). Sequences of the oligonucleotides are
given in Table I. A 20-µl binding reaction contained 10 µg of
nuclear extract, 1× binding buffer (10 mM Tris (pH 7.5),
50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 50% v/v glycerol), 30,000 cpm labeled probe,
100 ng of poly(dA-dT)·poly(dA-dT), and 50 ng of cold competitors
where indicated. Binding reactions were incubated for 20 min at room temperature and resolved on 5% polyacrylamide gels. Supershift assays
were performed as above except that a GKLF, BTEB2, SP1, or AML1
antibody (Santa Cruz, GKLF (T16x), BTEB2 (A-16x), AML1 (C-19x)) (2 µg/reaction) was added for 1 h before addition of the labeled probe.
Immunocytochemistry--
Adult rat VSMCs seeded at a density of
10,000 cells/well on 4-well chamber slides were allowed to attach and
were then serum-starved for 72 h before TGF-
1 or BMP2
treatment. Immunocytochemistry was carried out according to
manufacturer's instructions using a GKLF-specific or BTEB2 antibody
and a fluorescent-tagged anti-goat secondary antibody (Sigma).
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RESULTS |
Differential Expression of TGF-
s and BMPs in Rat Neonatal
Development--
Previously we demonstrated by Northern blotting that
a number of SM-marker genes including
-SM actin, calponin, SM-MHC,
and SM22
, were up-regulated during neonatal development (4). To confirm this expression pattern using RT-PCR, primers specific to
calponin, SM-MHC, and SM22
were used in PCR reactions with cDNA
derived from purified aortic VSMCs. This analysis confirmed up-regulation of SM markers between day 1 and week 2 of neonatal development (Fig. 1). Subsequently, the
same RT-PCR system was used to examine the developmental expression
profiles of TGF-
1-3, and the BMPs were identified above. All three
TGF-
isoforms displayed similar expression profiles and were
up-regulated during early neonatal development (neonatal day 1 to week
2) with high expression maintained until week 8 (Fig.
2). However, in the adult (week 12),
expression of TGF-
2 and -3, and to a lesser extent TGF-
1, declined (Fig. 2).

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Fig. 1.
SM-marker expression during VSMC
differentiation. RT-PCR was carried out using calponin-, SM-MHC-,
and SM22 -specific primers. Rat aortic VSMC cDNA samples were
from fetal day 17, fetal day 19, neonatal day 1, week 2, week 4, week
6, week 8, and adult. A, ethidium bromide stained agarose
gels of PCR products. B, graphs depicting relative mRNA
expression levels quantitated as described under "Experimental
Procedures." Data represent the mean ± S.D. of three separate
experiments.
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Fig. 2.
TGF- expression
during VSMC differentiation. RT-PCR was carried out using primers
specific to TGF- 1-3 and GAPDH. A, ethidium
bromide-stained gels of PCR products. B, graphs depicting
relative mRNA expression levels quantitated as described under
"Experimental Procedures." Data represent the mean ± S.D. of
three separate experiments.
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Before examining BMP expression profiles during development, we
determined which BMPs are expressed by adult rat medial VSMCs by using
degenerate oligonucleotides (Table I) designed to conserved BMP
sequences to amplify cDNA generated from these cells. This revealed
that BMP2-6, activin
A, and GDF5 (growth differentiation factor) (data not shown) are co-expressed by adult rat VSMCs. RT-PCR
profiling established that BMP2-7 and activin
A exhibit reproducible dynamic expression patterns during VSMC differentiation (Fig. 3). BMP5 and -7 expression was high
during fetal development and decreased during neonatal development. In
contrast, expression of BMP2, -3, -4, and -6 and activin
A was low
during fetal development, up-regulated during neonatal development,
and maintained at high levels until week 8. In the adult (week
12) expression of BMP4 and -6 declined, whereas expression of BMP2 and
-3 and activin
A remained elevated (Fig. 3). Although GDF-5 was
identified in the degenerate PCR screen, it was not possible to amplify
a GDF5 PCR product from these samples.

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Fig. 3.
BMP expression during VSMC
differentiation. RT-PCR was carried out using primers specific to
BMPs2-7, activin A, and GAPDH. A, ethidium bromide-stained
gels of PCR products. B, graphs depicting relative mRNA
expression levels quantitated as described under "Experimental
Procedures." Data represent the mean ± S.D. of three separate
experiments.
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Co-expression of Type I TGF-
47 BMP Receptors and Their Ligands
in Rat Neonatal Development--
Because TGF-
isoforms and BMPs
display distinct expression profiles during VSMC differentiation, we
examined whether the expression of TGF-
/BMP type I receptors was
also differentially regulated. This revealed that ALK2, -3, -5, and -6 and their ligands are co-expressed during VSMC differentiation (Fig.
4). The expression pattern of the BMP
receptor ALK6 was similar to that of BMP2, -3, -4, and -6, with the
greatest expression during neonatal development. ALK3, also a BMP
receptor, was expressed during the neonatal phase of development, but
its expression peaked specifically at week 2 (Fig. 4). In contrast
ALK5, a TGF-
receptor, and ALK2 an activin/BMP7 receptor were
expressed constitutively during development.

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Fig. 4.
ALK expression during VSMC
differentiation. RT-PCR was carried out using primers specific to
ALK 2, 3, 5, 6 and GAPDH. A, ethidium bromide-stained gels
of PCR products. B, graphs depicting relative mRNA
expression levels quantitated as described under "Experimental
Procedures." Data represent the mean ± S.D. of three separate
experiments.
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TGF-
s and BMPs Differentially Regulate
-SM Actin and SM22
mRNA Expression--
Co-expression of TGF-
1-3, BMP2-7, and
their receptors during VSMC development suggested a putative role for
these factors in the regulation of VSMC phenotype. TGF-
1 and activin
A have been shown to up-regulate
-SM actin gene expression (9, 38), but the effects of TGF-
2, TGF-
3, and the BMPs on VSMC
differentiation marker gene expression are unknown. We chose to
evaluate the effects of BMP2, -4, and -6 because these BMPs were made
available to us and are expressed in normal adult vasculature.
Confluent adult rat VSMCs were growth-arrested and treated with TGF-
(2.5 ng/ml) or vehicle for 48 h. All three TGF-
isoforms increased SM22
and
-SM actin mRNA expression relative to
vehicle-treated VSMCs (Fig.
5A). In contrast, BMP2 and -6 (50 ng/ml) down-regulated
-SM actin and SM22
expression (Fig.
5B), whereas BMP4 up-regulated expression levels of these
genes relative to vehicle-treated cells (Fig. 5B).

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Fig. 5.
TGF- s and BMPs
reciprocally regulate smooth muscle marker mRNA expression.
Confluent growth arrested adult rat VSMCs were treated with TGF- 1-3
(2.5 ng/ml) or vehicle or BMP-2, -4, and -6 (50 ng/ml) or vehicle.
After 48 h, RNA was extracted and used for Northern analysis.
Shown are autoradiographs of northern blots from TGF- isoform- and
vehicle-treated RNA samples (A) and BMP- and vehicle-treated
RNA samples (B) hybridized with -SM actin or SM22 .
Blots were re-hybridized using 18 S for normalization.
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TGF-
-Superfamily Members Affect Smooth Muscle-specific Protein
Expression--
Western blotting revealed that the effects of
TGF-
1-3 and BMP-2, -4, and -6 protein expression were consistent
with their effects on
-SM actin mRNA expression. TGF-
1-3
up-regulated
-SM actin protein expression (Fig.
6A). In contrast BMP2 and -6 down-regulated and BMP4 up-regulated expression of
-SM actin and
another SM differentiation marker, calponin (Fig. 6B). The
level of expression of SM-MHC protein was also analyzed but was too low
to detect (not shown).

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Fig. 6.
Effects of
TGF- -superfamily members on smooth muscle
marker protein expression. Confluent growth arrested adult rat
VSMCs were treated with TGF- 1, -2, -3 (2.5 ng/ml), BMP2, -4, -6 (50 ng/ml), or vehicle either alone or in the presence of cyclohexamide (10 µg/ml) for 48 h. Duplicate gels were silver-or Coomassie
Blue-stained to confirm equal sample loading. A, Western
blot showing -SM actin increases in cells treated with TGF- 3 as
compared with vehicle. This increase does not occur in the presence of
cyclohexamide. The results obtained from TGF- 1 and -2 were similar
to those for TGF- 3 and are not shown. B, Western blots
demonstrating differential effects of BMPs on SM-marker protein
expression in VSMCs. These effects are inhibited in the presence of
cyclohexamide (10 µg/ml) (C).
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To determine whether the observed changes in protein levels require new
protein synthesis, VSMCs were treated with TGF-
-superfamily members
for 48 h in the presence of cyclohexamide (10 µg/ml). Cyclohexamide treatment blocked the effects of the TGF-
-superfamily members on
-SM actin protein expression, indicating that changes in
protein levels are dependent on new protein synthesis (Fig. 6,
A and C).
The TCE Element Is Required for BMP Responsiveness of the SM22
Promoter--
Transient transfections were carried out using SM22
promoter constructs p80 and p55 linked to a luciferase reporter (Fig. 7A) (36). The p80 construct
spanned bases
303 to +65 and has been shown to be 7-fold more active
in VSMCs than the longer p55 construct (
1515 to +65) and two shorter
(
193 to +65 and
117 to +65) constructs (36). The longer p55
construct was less active than p80 presumably due to upstream silencer
sequences (36).

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Fig. 7.
Transient transfection analysis. VSMCs
were transiently transfected with the p80 ( 303 to +65) and p55
( 1515 to +65) SM22 promoter luciferase reporter constructs and
relative luciferase activity was measured after 48 h.
A, sequence of the p80 construct and location of the TCE and
other identified consensus binding sites. The sequence of the mutated
TCE element is given below the wild type sequence (boxed).
B, using the p55 SM22 promoter construct significant
activation of promoter activity by TGF- 1 and significant repression
by BMPs was observed. C, relative luciferase activity in
cells transfected with the TCE wild type (black) or TCE
mutated (white) p80 construct. Note significant activation
(with TGF- 1) and repression (with BMPs) in the presence of a wild
type TCE only. Vehicle controls are shown in gray. In
the presence of vehicle control, no difference was seen in basal
activity between the wild type and mutant p80 constructs. D,
after transfection with the wild type p80 SM22 promoter, construct
cells were treated with TGF- 1 alone or in combination with BMP-2,
-4, and -6. The relative luciferase activity of the TGF- 1-treated
samples was set to 1.0. Co-treatment with BMPs attenuated the effects
of TGF- 1 treatment. All results represent the mean of 3-6
experiments performed in triplicate ±S.E. (* = p < 0.05). Abbreviations are as follows: V, vehicle;
T1,TGF- 1; B2, BMP2; B4, BMP4;
B6, BMP6.
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Transactivation assays with both the p55 luc and p80 luc promoter
constructs revealed that BMP-2, -4, and -6 all repress SM22
promoter
activity in VSMCs (Fig. 7, B and C).
Down-regulation of SM22
promoter activity by BMP2 and -6 correlates
with the effects of these ligands on SM-marker expression at the
mRNA and protein levels (Figs. 5 and 6). Down-regulation of SM22
promoter activity by BMP4 contrasts with the effects of BMP4 on SM22
mRNA and protein levels (Figs. 5 and 6), suggesting that other
promoter elements upstream of those included in the longer p55
construct are required for BMP4 enhancement of SM22
promoter
activity. This contrasted with TGF-
1, which enhanced SM22
promoter activity of both the p55 an p80 constructs.
To determine whether the TCE element is required for BMP responsiveness
of the SM22
promoter, the TCE element was mutated. Mutation of the
TCE element abolished BMP-induced repression (Fig. 7C),
indicating that an intact TCE element is required for BMP-regulated SM22
promoter repression.
To address whether the BMPs can modulate TGF-
1-induced SM22
promoter activity, the wild type SM22
p80 promoter construct was
transfected in VSMCs followed by treatment with TGF-
1 in combination
with BMP2, -4, or -6. These studies demonstrated that BMP-2, -4, and -6 can negatively modulate TGF-
1-induced increases in SM22
promoter
activity (Fig. 7D).
TGF-
1 and BMPs Enhance SMC Nuclear Protein Binding to a
Cis-regulatory TCE--
EMSAs performed with nuclear proteins prepared
from VSMCs treated with TGF-
1 or BMP2, -4, or -6 all formed three
specific shifted complexes, c1-c3, with the TCE probe (Fig.
8A). Although c1-c3 were also
apparent in assays with proteins from vehicle-treated cells, a greater
degree of c1-c3 formation was observed in experiments utilizing nuclear
protein extracts derived from cells treated with TGF-
-superfamily
members (compare lane 2 to lanes 5 and 8). Formation of these complexes was competed out by the
addition of excess cold wild type TCE competitor. Excess of a mutated
TCE cold competitor did not affect formation of the three complexes (Fig. 8A).

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Fig. 8.
Analysis of TCE binding proteins.
A, a competitive EMSA showing the effects of TGF- 1 and
BMP2 treatment on protein complex formation on the TCE of the SM22
promoter. Nuclear extracts were obtained from adult rat VSMCs treated
with vehicle (V), TGF- 1 (T1), or BMP2
(B2). Competitors were added to the gel shift reactions as
follows. Lanes 2, 5, and 8, no
competitor DNA; lanes 3, 6, and 9, 50 ng of wild type double-stranded competitor DNA; lanes 4 and
7, 10- 50 ng of mutant double-stranded competitor DNA.
Shifted complexes c1-c3 are marked. B, supershift analysis
to determine proteins binding in complexes c1-c3. Nuclear extracts from
above were used in supershift reactions containing the following
additional antibodies. , no antibody control; B, BTEB2;
G, GKLF; A, AML1. Shifted complexes are labeled
c1-c3. Lanes 2, 6, and 10, no
antibody; lanes 3, 7, and 11, BTEB2
antibody; lanes 4, 8, and 12, GKLF
antibody; lanes 5, 9, and 13, AML1
antibody.
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KLF4 Is the Predominant Protein in TCE Complexes c1 and
c2--
The TCE has been shown to bind Sp1 and the Krüppel-like
transcription factors BTEB2 (KLF5) and GKLF (KLF4) (12). To determine whether these proteins are constituents of the VSMC TCE binding complexes, supershift assays were performed with antibodies specific for Sp1, BTEB2, and GKLF. An AML1 antibody was used as a negative control (Fig. 8B). Of the three specific complexes formed,
the two upper complexes, c1 and c2, were competed out by the
GKLF-specific antibody in the control TGF-
1- and BMP2-treated
nuclear extracts (lanes 4, 8, and 12).
No supershift or competition was observed with the Sp1 (not shown),
BTEB2, or AML1 antibodies, thus identifying GKLF and an unknown lower
migrating protein complex (c3) as the predominant TGF-
1- and
BMP-induced protein that binds to the TCE.
Members of the TGF-
Superfamily Induce the Expression of a
Shorter Isoform of GKLF/KLF4--
Western blotting revealed
that after treatment of VSMCs with TGF-
1, BMPs, and activin A, a
lower molecular mass GKLF isoform was induced (Fig.
9A). A single 52-kDa GKLF
protein was observed with the control extract, and a doublet containing
an additional smaller GKLF protein was observed in extracts from cells
treated with TGF-
-superfamily members. It is possible that the lower molecular mass GKLF protein is a component of the c2 complex induced by
TGF-
1 or BMP2 shown in Fig. 8; however, further characterization of
the c2 complex is required before definitive conclusions can be drawn.
Further analysis excluded phosphorylation and acetylation as the cause
of the size differences between the GKLF protein bands (not shown).

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Fig. 9.
GKLF induction by
TGF- -superfamily members. A,
Western blot showing the induction of GKLF after treatment of rat VSMCs
with members of the TGF- superfamily. Shown are vehicle (lane
1)-, TGF- 1 (lane 2)-, BMP2 (lane 3)-,
BMP4 (lane 4)-, BMP6 (lane 5)-, BMP7 (lane
6)-, and activin A (lane 7)-treated nuclear extracts
respectively. B, immunocytochemistry showing the effect of
TGF- and BMP2 on endogenous levels of GKLF/KLF4 expression. Panels
depict control, TGF- 1-, and BMP2-treated adult rat
VSMCs.
|
|
Immunocytochemical assays further demonstrated the induction of
GKLF/KLF4 by TGF-
1 and BMP2 above the levels in vehicle-treated cells (Fig. 9B). In contrast, BTEB2 expression was not
observed in treated or control adult rat VSMCs (not shown).
 |
DISCUSSION |
In this study we demonstrated that multiple TGF-
-superfamily
members can modulate expression of VSMC phenotypic markers via a common
cis-acting TCE element and that BMPs are able to modulate the effect of TGF-
1 on the transactivation of a SM-specific gene, SM22
. Modulation of SM-marker expression, whether positive or negative, was associated with the induction of a smaller GKLF/KLF4 protein that binds to the TCE element, a sequence common to a number of
SM-specific gene promoters. In vivo TGF-
-superfamily members, which are important modulators of VSMC phenotype, are differentially co-expressed during fetal/neonatal to adult development. This suggests that the ratio of different family members during development is important for VSMC phenotypic change and stability. It
follows that a shift in the expression ratios of the different family
members may act as a catalyst in the induction of vascular disease.
Recently, transgenic mice bearing an SM22
-lacZ promoter construct
with a mutated TCE element revealed that the TCE element is essential
for the in vivo expression of the SM-marker SM22
(12). In
our system BMP-2, -4, and -6 were shown to transcriptionally repress
SM22
promoter activity. The effects of BMP2 and -6 were consistent
with results seen at the mRNA level; however, BMP4 repression of
SM22
promoter activity was in contrast to its induction of SM22
mRNA. The difference between the transcriptional reporter assays
and the mRNA levels is probably due to the presence of unidentified
enhancer elements not present in the promoter regions covered by the
p80 and p55 reporter constructs.
Utilization of a p80 reporter construct containing a mutated TCE
element established that an intact TCE element is required for
BMP-modulated SM22
promoter repression. The discovery of TCE-dependent BMP-modulated SM-marker expression suggested
that the BMPs might compete with TGF-
1. This was validated in
vitro as co-treatment of VSMCs transfected with the p80 reporter,
with TGF-
1, and BMP2, -4, or -6 showed that each of these BMPs
decreased TGF-
1-induced SM22
promoter activity, emphasizing the
importance of the expression levels of multiple TGF-
-superfamily
members in the regulation of VSMC phenotype.
Both positive and negative regulators of SM22
promoter activity
induced TCE binding of VSMC nuclear proteins. Based on work by Adam
et al. (12), we speculated that TGF-
-superfamily members may induce binding of different Krüppel-like transcription
factors to the TCE. Adam et al. (12) identify GKLF as a
TCE-binding protein using yeast one-hybrid analysis and showed that
GKLF expression is down-regulated by TGF-
1. Co-transfection assays
in C3H10T1/2 cells revealed that GKLF-repressed
TGF-
1 induced
-SM actin and SM22
promoter activity, whereas
another Krüppel-like transcription factor, BTEB2/KLF5, activated
the SM22
promoter in this cell type (12). Although it is possible
that such a reciprocal mechanism may exist in vivo, we were
unable to detect BTEB2 binding by EMSA after TGF-
1 or BMP2 treatment
nor did we detect BTEB2 protein by Western blotting or
immunocytochemistry. Of particular note, the BTEB2 antibody used by
Adam et al. (12) in the EMSA analysis was likely to have
cross-reacted with other KLF family members because 10 of the 12 amino
acids of the peptide used to generate it are identical to GKLF (12).
BTEB2 was originally identified as a marker of dedifferentiated VSMCs
because it activates transcription of SMemb/nonmuscle myosin
heavy chain-B and has been shown to have a role in vascular remodeling
after injury (39). It is highly expressed during vascular development
but is down-regulated in adult vessels; therefore, its absence in
cultured adult VSMCs in serum-free conditions is not unexpected.
In this study we identified a shorter GKLF variant that was induced
after TGF-
and BMP2 treatment. It is likely that the different GKLF
variants are the result of post-translational modification of GKLF;
however, this difference was not due to phosphorylation nor
acetylation. GKLF has previously been shown to contain both activation
and repression domains (40). Because both positive and negative
regulators of SM markers induce the smaller variant, it is likely that
additional trans-acting factors are involved in SM-promoter regulation.
These additional factors may bind directly to the GKLF complex or to
other cis elements within SM-promoters.
In addition to defining a downstream target of TGF-
- and
BMP-modulated transcriptional regulation in VSMCs, this is the first report to describe the expression patterns of TGF-
-superfamily type
I receptors and their ligands during neonatal VSMC differentiation. The
dynamic in vivo expression profiles of these family members and their receptors further supports their role as major effectors of
VSMC phenotype. TGF-
isoforms displayed similar expression patterns
during neonatal development; however, expression of TGF-
2 and
TGF-
3 declined in the adult. Isoform-specific differences in
expression have previously been described in rat medial VSMCs 48 h
after balloon injury, where TGF-
1 and -3 mRNA expression was
elevated, whereas TGF-
2 mRNA expression declined (41). Although
TGF-
isoforms have similar expression patterns and effects on
SM-marker expression, it is possible that they differentially regulate
other processes such as fibrogenesis or cell proliferation (42).
BMP5 and -7 were maximally expressed during fetal development, whereas
BMP2, -3, -4, and -6 were up-regulated during neonatal development.
Because little is known about the effect of the BMPs on VSMCs, it is
difficult to speculate from these expression profiles as to the role of
each factor. During neonatal development, a number of significant
changes occur in VSMCs, including increased extracellular matrix,
SM-contractile protein expression, and decreased proliferation (5).
Recently, the effects of BMP2 and BMP7 have been examined in injured
arteries (20, 43). BMP7 maintained VSMC phenotype in occluded rat renal
arteries, and BMP2 was shown to inhibit VSMC proliferation after
balloon injury to rat carotid arteries (26, 43). We showed that BMP2
expression increased during the neonatal phase of development, a time
when VSMC proliferative index decreases. SM markers are up-regulated
in vivo during this phase, yet in vitro BMP2
down-regulated SM-marker expression. Thus, BMP2 may regulate other
aspects of VSMC biology that alter with differentiation, such as
proliferation, further supporting the notion that a balance of positive
and negative regulators determines VSMC phenotype (5).
In summary, this study has provided mechanistic insight into BMP
modulation of VSMC phenotype. Further studies into the roles played by
specific BMPs in regulating VSMC phenotype are now required, given the
correlation of VSMC phenotypic modulation with vascular disease.