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INTRODUCTION |
Vascular cell adhesion molecule-1
(VCAM-1)1 is a member of the
immunoglobulin superfamily first identified as an adhesion protein expressed on cytokine-activated endothelial cells (1-3). Through its
interaction with its integrin counter receptor, very late antigen-4
(VLA-4), it mediates cell-cell interactions, which have been shown to
be involved in recruitment of mononuclear leukocytes to the vascular
lesions observed in early atherosclerosis. VCAM-1 and VLA-4 are also
implicated during skeletal muscle myogenesis and developing central
nervous system on neuroepithelial cells in embryogenesis (4-6).
In the vascular system, VCAM-1 is expressed in human SMCs during
embryonic development and in atherosclerotic plaques, and it
colocalizes with VLA-4 (7, 8). VCAM-1-deficient mice embryos have been
shown to have an abnormal placental development in which the allantois
failed to fuse to the chorion, and they displayed abnormalities in
their developing hearts (9, 10). In vitro, VCAM-1 is induced
in SMCs after cytokine stimulation (11, 12). It is also coinduced with
SMC marker proteins during SMC differentiation in vitro, and
treatment with antibodies against VCAM-1 or its ligand VLA-4 has been
found to interfere with the expression of mRNA of smooth
muscle-specific markers during redifferentiation (8). Thus, VCAM-1
seems to be expressed and involved during smooth muscle
differentiation. Investigation of mechanisms controlling its expression
should provide some insight into specific gene regulation in vascular
smooth muscle during differentiation.
Studies on the activity of the VCAM-1 gene promoter have shown that in
endothelial cells, TNF-
induction of VCAM-1 is dependent on two
adjacent
B sites located at positions
77 and
63 relative to the
single transcriptional start site (13, 14). These
B elements are
also important for VCAM-1 induction in the embryonic carcinoma cell
line P19 stimulated to differentiate along a neural pathway by
treatment with retinoic acid (6). Thus, these previous works suggested
that NF-
B proteins could be involved in the VCAM-1 expression in
SMCs. NF-
B proteins belong to the Rel protein family, transcriptional regulatory proteins, including p65 (RelA), RelB, p52/100 (NF-
B1), p50/p105 (NF-
B2), v-Rel, and the product of the
c-rel proto-oncogene (15-19). Interestingly, Bellas
et al. (20) have described a novel NF-
B member termed
SMC-Rel, essential for proliferation of cultured bovine vascular SMCs.
In most unstimulated cell types, NF-
B is sequestered in the
cytoplasm complexed with an inhibitory protein, I-
B, in a
non-DNA-binding form (21). Stimulation of cells with cytokines (22) and
several other agents (23) results in the release of NF-
B complex
from I-
B and its translocation into the nucleus, where it
transcriptionally regulates the expression of a wide variety of genes
through specific
B elements (23). The activity of the NF-
B/Rel
family is controlled at different levels, e.g. by the
different combination of NF-
B/Rel dimers formed, and the sequence of
B sites recognized by them.
In this study, the role of NF-
B in VCAM-1 expression during SMC
differentiation was compared with that induced by TNF-
. Electrophoretic mobility shift assay (EMSA) failed to detect any induction of NF-
B/Rel protein binding activity on
B binding sites
on the human VCAM-1 gene promoter in differentiated SMCs, although it
was observed in TNF-
-activated SMCs. Addition of the antioxidant
pyrrolidine dithiocarbamate (PDTC), inhibitor of NF-
B/Rel activity
(23, 24), did not modify VCAM-1 mRNA induction during SMC
differentiation, in contrast with the strong inhibition of
TNF-
-induced expression of VCAM-1. These results indicate that
NF-
B/Rel proteins are not involved in VCAM-1 gene expression during
SMC differentiation.
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MATERIALS AND METHODS |
Cytokines and Antibodies--
Recombinant human TNF-
was
provided by Genzyme, Inc. (Cambridge, MA). Murine monoclonal antibody
directed against human VCAM-1 (monoclonal antibody 1G11, diluted 1:50)
was purchased from Immunotech. Anti-human smooth muscle myosin heavy
chain (sm-MHC) monoclonal antibody was kindly provided by Dr. M. Glukhova (25) (diluted 1:100). Rabbit polyclonal antibodies to
NF-
B/rel family member p50 and p65 were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA; diluted 1:200).
Cell Culture--
SMCs were isolated from media of human
thoracic aortas by enzyme digestion as described previously (26). SMCs
were cultured in Ham's F-10 medium (Life Technologies, Inc.)
supplemented with 5% fetal calf serum, 5% heat-inactivated human
serum, 5 mM HEPES, 50 units/ml penicillin, and 50 mg/ml
streptomycin, at 37 °C in a 5% CO2, 95% air
atmosphere. Studies were conducted on SMCs at passage two, seeded at a
high seeding density (5 × 104 cells/cm2).
In order to obtain the different phenotypes of SMCs (undifferentiated or differentiated), we used various culture conditions (8, 27, 28). At
confluence, the cells were either maintained undifferentiated in medium
supplemented with serum, or induced to differentiate in a defined
serum-free medium containing a 1:1 mixture of Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) and Ham's F-10 plus insulin
(10
6 M, Sigma), transferrin (200 µg/ml,
Sigma), ascorbate (0.2 mM, Sigma), and sodium selenite
(6.25 ng/ml, Sigma). These conditions designed to induce
redifferentiation were usually maintained for 6 days. However, for
time-course or inhibition experiments, as some smooth muscle markers
and VCAM-1 mRNA were rapidly induced within day 1 in defined
serum-free medium (8), cells were used 1, 6, or 24 h after the
switch of medium.
In order to obtain a VCAM-1 cytokine-induced expression in the absence
of any redifferentiation process, confluent SMCs maintained for 6 days
in serum-supplemented medium, were treated with TNF-
(1000 units/ml)
24 h before analysis.
In order to inhibit NF-
B activity, cells were treated with PDTC
(Sigma) at a final concentration of 100 µM. Initially the confluent cells were pretreated for 1 h with PDTC, and then
maintained in culture for an additional 24 h in the presence of
PDTC and either TNF-
in medium plus serum or in serum-free medium.
Cells maintained in serum and treated for 25 h with PDTC were used
as control. The effect of PDTC on SMCs viability was assessed by trypan
blue exclusion; less than 5% cell mortality was observed. The well
plates were rinsed with phosphate-buffered saline (PBS), and RNAs were
extracted and analyzed by RT-PCR.
Immunocytochemistry and Immunofluorescence--
SMCs were grown
in Lab-Tek two-well chambers (Nunc Inc., Naperville, IL), then rinsed
with PBS, fixed, and permeabilized with cold methanol for 10 min. Cells
were incubated with primary and secondary antibodies in BSA/PBS 1% for
1 h. For sm-MHC detection, the secondary antibody, a
biotin-conjugated anti-mouse IgG from sheep (Amersham Pharmacia
Biotech, diluted 1:100), and the streptavidin-biotinylated horseradish
peroxidase complex (RPN 1051, Amersham Pharmacia Biotech, diluted
1:100) were applied. For NF-
B/Rel family protein labeling, the
secondary antibody, a biotin-conjugated, anti-rabbit donkey IgG
(diluted 1:100), and the streptavidin-Texas Red complex (RPN 1223, Amersham Pharmacia Biotech, diluted 1:100) were used.
Flow Cytometry--
For flow cytometric analysis, monodispersed
suspensions of SMCs were prepared by brief incubations in buffer (6 mM glucose, 5.3 mM KCl, 125 mM
NaCl, 18 mM Hepes, 1× PBS, and 0.2 mM EDTA) at
37 °C. The cells were then incubated with 50 µl of monoclonal antibody against human VCAM-1 in 2% BSA/PBS for 30 min in ice. After
washing, cells were treated with sheep anti-mouse Ig,
fluorescein-linked whole antibody (Amersham Pharmacia Biotech) for 30 min in ice, washed three times, and resuspended in 500 µl of PBS. The
samples were analyzed in a Coulter type XL flow cytometer.
RNA Analysis--
Total cellular RNA was prepared using a
single-step acid guanidium isothiocyanate/phenol/chloroform extraction
method (29): first-strand cDNA synthesis by reverse transcription
(30) using 1 µg of total RNA and 20 µg/ml oligodeoxythymidine and
200 units of Moloney murine leukemia virus RNase H-reverse
transcriptase (Life Technologies, Inc.) with incubation for 1 h at
42 °C. Specific primers for G3PDH, VCAM-1, and sm-MHC were used as
described previously (8) to amplify regions of cDNA copies from
total RNA.
RT-PCR Product Analysis--
The samples were analyzed on 1.5%
agarose gels. In order to verify the specificity of generated PCR
products, hybridizations were performed with specific probes for VCAM-1
and sm-MHC, or with an internal oligonucleotide for G3PDH after
Southern transfer (31). Probes for human VCAM-1 were synthesized by
RT-PCR and cloned in pBluescript vector (Stratagene) as described
previously (12). For human sm-MHC probe, human sm-MHC cDNA was
amplified by PCR for 30 cycles at 60 °C for annealing using primers
as described elsewhere (8). The PCR product obtained was purified,
digested in a 392-bp fragment, and subcloned into pBluescript plasmid
according to standard procedures. The specificity of the PCR product
was ensured by the DNA sequence determined by the dideoxy
chain-termination method with modified T7 DNA polymerase. The VCAM-1
and sm-MHC cDNA fragments were labeled by random priming with the
kit Ready-To-Go (Biolabs). Hybridization was done at 42 °C overnight
in the presence of 106 cpm/ml in buffer 50% (v/v)
formamide, 4× SSC, 10× Denhardt's solution (1× Denhardt's solution
is 0.02% polyvinyl pyrrolidone, 0.02% Ficoll, 0.02% BSA), 0.1% SDS,
and sheared denatured salmon sperm DNA at 0.4 mg/ml. Filters were
washed in 2× SSC for 10 min at room temperature, followed by two
washes in 0.1× SSC for 30 min at 55 °C. Filters were exposed to
Kodak XAR-5 film with an intensifying screen.
Preparation of Nuclear Extracts--
Preparation of nuclear
extracts was based on the methodologies described by Li and Schreiber
(32, 33). The cultured cells (approximately 2 × 107
cells) were washed with cold PBS and harvested after trypsin treatment.
The cells were centrifuged at 4,000 rpm for 10 min, and the cell pellet
was resuspended in 800 µl of cold buffer A (10 mM HEPES,
pH 8, 50 mM NaCl, 0.5 M sucrose, 1 mM EDTA, pH 8, 0.5 mM spermidine, 0.15 mM spermine). The cells were allowed to swell on ice for 15 min, then 25 µl of a 10% solution of Nonidet P-40 was added to
release the nuclei. Nuclei were recovered by centrifugation at 3,000 rpm for 10 min at 4 °C. The nuclear pellet was resuspended in 500 µl of buffer B (10 mM HEPES, pH 8, 50 mM NaCl, 25% glycerol, 0.1 mM EDTA, pH 8, 0.5 mM
spermidine, 0.15 mM spermine) in the presence of protease
inhibitors (pepstatin, leupeptin, aprotinin, antipain, and chymostatin
at 5 µg/ml, 1 mM Pefabloc, 2 mM benzamidine)
and 1 mM dithiothreitol. The nuclear pellet was recovered
by centrifugation, and nuclear proteins were extracted by incubation
for 1 h at 4 °C in 100 µl of buffer C (the same as buffer B
except 400 mM NaCl). The nuclear extract was centrifuged at
13,500 rpm for 40 min, and the supernatant was dialyzed three times
against 250 ml of buffer D (20 mM HEPES, pH 8, 50 mM KCl, 20% glycerol, 0.2 mM EDTA, pH 8, 1 mM dithiothreitol, 2 mM benzamidine) for 1 h. Nuclear extracts were stored in aliquots at
80 °C. Protein
concentration was measured by the Bradford assay (Bio-Rad kit)
(34).
Oligonucleotide Sequences--
In order to prepare
double-stranded oligonucleotides containing the upstream
B elements
of human VCAM-1 gene, synthetic complementary single-stranded
oligonucleotides were annealed and then purified by polyacrylamide gel
electrophoresis. Gel-purified double-stranded oligonucleotides were
labeled on their 5' ends with [
-32P]ATP (5000 Ci/mmol)
(Amersham Pharmacia Biotech) using T4 polynucleotide kinase (Life
Technologies, Inc.) and used as probes in EMSA. Oligonucleotides were
chosen according to Iademarco et al. (13). Oligonucleotide
B corresponds to the two wild-type
B sites (boldface) taken from
the DNA sequence located at bp
77 to 63 of the VCAM-1 gene promoter:
CTGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCGCCT. Oligonucleotide
BM was identical to
B wild type sites except for
4 nucleotide mutations (underlined) in each of the two NF-
B-DNA binding sites according to Marui et al. (35):
CTGCCCTGAGTCACGCCTTGAAGAGACATCACTCCGCCT. An oligonucleotide (YY1) corresponding to a probe for another transcription factor was used in competition experiments to ensure the
specific
B DNA binding activity with NF-
B proteins: TCTGTCTCCATTTTTTCTCT.
Electrophoretic Mobility Shift Assay--
EMSA were performed
with 25 µl of binding mixture containing 1 µg of double-stranded
poly(dI-dC), 40 fmol of 32P-labeled annealed
B
oligonucleotides, 8 µg of nuclear extract in 12 mM HEPES,
pH 8, 50 mM KCl, 12% glycerol, 1 mM
MgCl2, 0.6 mM dithiothreitol, 0.12 mM EDTA, 1.2 mM benzamidine. Reactions were
incubated at room temperature for 30 min, and DNA-protein complexes
were resolved by non-denaturing polyacrylamide electrophoresis in 5%
acrylamide, employing 0.022 M Tris borate, 0.5 mM EDTA, pH 8, as buffer. Following electrophoresis, gels
were dried and DNA-protein complexes were localized by autoradiography.
Competition studies were performed by adding unlabeled double-stranded
oligonucleotides to the binding reaction.
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RESULTS |
VCAM-1 Induction Model during SMC Differentiation--
In order to
determine the role of the NF-
B/Rel protein family in VCAM-1
expression in SMCs, we used an in vitro model of SMC
phenotypic modulation. Concerning the modulation of differentiation process, we analyzed the protein and mRNA levels of sm-MHC, a marker of the fully differentiated phenotype of SMCs (36-38). In 6-day
post-confluent cells, maintained in medium with serum, a faint
immunostaining for sm-MHC was observed, suggesting a dedifferentiated phenotype (Fig. 1, S). When
these post-confluent cells in medium with serum were stimulated for
24 h with TNF-
, the same low expression of sm-MHC was observed
(Fig. 1, T), showing the lack of effect of TNF-
on SMC
differentiation. On the other hand, when the confluent cells were
cultured for 6 days in the defined medium without serum, a strong
immunostaining for sm-MHC was noted, indicative of redifferentiation (Fig. 1, D). Analysis of mRNA content by RT-PCR and
Southern transfer revealed a low level of sm-MHC mRNA in cells
cultured in medium with serum that was stimulated or not stimulated
with TNF-
(Fig. 2). In SMCs cultured
in defined serum-free medium, a high level of sm-MHC mRNA was
detected (Fig. 2). In this in vitro model, VCAM-1 expression
showed a different pattern to that of sm-MHC. Flow cytometry and RT-PCR
analysis showed that SMCs maintained in medium with serum had a low
level of VCAM-1 protein and mRNA (Table
I and Fig. 2). However, administration
for 24 h with TNF-
or cultivation in defined serum-free medium
for 6 days induced an increase in protein and mRNA content of
VCAM-1 (Table I and Fig. 2). These results showed that VCAM-1
expression was increased in two conditions, either by cytokine
treatment in SMCs expressing low levels of sm-MHC, or by medium switch
inducing redifferentiation characterized by a high level of sm-MHC
expression. These results suggested that VCAM-1 was induced in two
different cell populations, in TNF-
-stimulated undifferentiated SMCs
and in redifferentiated SMCs.

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Fig. 1.
sm-MHC protein expression in an in
vitro model of SMC phenotypic modulation. Confluent
second-passaged SMCs cultured in the presence of serum (S),
stimulated by TNF- (T), or maintained in defined medium
without serum (D) were fixed with cold methanol and labeled
immunocytochemically with a monoclonal antibody directed against human
sm-MHC. A negative control was obtained by omitting the primary
antibody (C, control). The nuclei were stained by Hemalun.
Original magnification, ×20. The high expression of sm-MHC
(D) indicated that the SMC redifferentiate in defined
serum-free medium.
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Fig. 2.
Southern blot analysis of VCAM-1 and sm-MHC
mRNA expression in SMC redifferentiation model. One µg of
total RNA obtained from post-confluent SMCs cultured in medium
supplemented with serum for 6 days, activated (T) or not
(S) with 1000 units/ml TNF- for 24 h, and from
post-confluent SMCs cultured in a defined serum-free medium for 6 days
(D) were used for RT-PCR analysis. Ten µl of DNA amplified
product were analyzed in 1.5% agarose gel and transferred on
nitrocellulose membrane. Hybridizations with specific end-labeled
probes were used to ensure specificity of amplified products. DNA
probes are described under "Materials and Methods." G3PDH, used as
internal control, VCAM-1, and sm-MHC mRNA expression were analyzed
in the three conditions.
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Table I
VCAM-1 protein expression in the in vitro model of SMC
redifferentiation
Human undifferentiated serum-cultured SMCs (Serum), undifferentiated
TNF- -stimulated SMCs (TNF- ), and redifferentiated SMCs (Defined
serum-free medium) were immunolabeled with anti-VCAM-1 monoclonal
antibody (1G11). A fluorescent secondary antibody was applied, and
5,000 cells were analyzed by flow cytometry. Mean fluorescence obtained
with no antibody was subtracted from the values obtained with specific
antibody. Values are means of three independent experiments.
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Localization of NF-
B/Rel Proteins during VCAM-1 Induction on
SMCs--
In order to determine the cellular distribution of
NF-
B/Rel proteins in SMCs, the immunostaining of the p50 and p65
subunits of NF-
B complex was studied in SMCs cultured under
different conditions at 6 days (Fig.
3a). p65 and p50 were found in
the nuclei of both undifferentiated SMCs cultured in serum and in TNF-
-treated SMCs (Fig. 3a, S and
T, respectively). However, in contrast with the strong
labeling of the cytokine-stimulated nucleus, a fainter nuclear staining
and a persistent cytoplasmic labeling was observed in cells maintained
only in medium with serum. In contrast, only a cytoplasmic labeling of
the p50 and p65 subunits with no nuclear staining was observed in
differentiating SMCs cultured in defined serum-free medium (Fig.
3a, D). This suggested that NF-
B proteins were
translocated to the nucleus when SMCs were cultured in medium
supplemented with serum or stimulated by TNF-
. This p50 and p65
protein translocation was not observed in redifferentiated SMCs.

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Fig. 3.
NF- B protein distribution in cultured SMCs
during redifferentiation process. Cells were stained with rabbit
polyclonal anti-p50 or anti-p65 antibody. The secondary antibody, a
biotin-conjugated anti-rabbit donkey IgG, was linked with
streptavidin-Texas Red complex. a, a p50 and p65 subunit
localization in culture model was studied. Confluent SMCs were cultured
for 6 days in serum-supplemented medium (S and T)
or in serum-free medium (D). In contrast with the strong
nuclear labeling observed in TNF- -stimulated SMCs (T),
only the cytoplasm was seen to be labeled in defined serum-free medium
(D). A faint nuclear staining is noted in SMCs maintained in
serum (S). b, a time course of p50 subunit was
performed. Confluent SMCs were cultured for 1, 6, or 24 h in
serum-supplemented medium without (S) or with TNF-
(T), or in serum-free medium (D). Antiserum
to p50 was used to immunostain SMCs. After TNF- stimulation
(T), a positive nuclear labeling was evident after 6 h
of stimulation and maintained for 24 h. In redifferentiating SMCs
in serum-free medium (D), only cytoplasmic staining was
observed at any time. Original magnification, ×20.
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In order to exclude an early involvement of NF-
B system in VCAM-1
expression during SMC redifferentiation process, we analyzed the time
course of p50 Rel protein translocation in our culture model (Fig.
3b). As the result obtained after 6 days, we observed a weak
p50 subunit staining into nucleus of SMCs maintained in serum. After
TNF-
stimulation, a slight p50 nuclear staining was evident at
1 h (Fig. 3b, T). A maximal positive nucleus
labeling was seen after 6 h of TNF-
stimulation and was
maintained for 24 h (Fig. 3b, T). In
redifferentiated SMCs, maintained in serum free-medium, we did not
observe any nucleus staining but only a cytoplasmic labeling (Fig.
3b, D). These results seemed to confirm the
non-involvement of NF-
B proteins in redifferentiated SMC VCAM-1 expression.
Characterization of Binding Activity of NF-
B/Rel Proteins during
VCAM-1 Induction on SMCs--
EMSA was employed in an attempt to find
out whether nuclear proteins interact specifically with
B elements
of the VCAM-1 gene promoter. Nuclear extracts of SMCs harvested in the
three culture conditions described above were incubated with a
double-stranded end-labeled oligonucleotide probe extending from
position
88 to
56 of the human VCAM-1 promoter. As shown in Fig.
4, the nuclear extract of
undifferentiated SMCs cultured in medium with serum (S)
formed one DNA-protein complex (B1) with the probe. The complex was
competed with a 10-50-fold molar excess of unlabeled
B
oligonucleotide, whereas addition of a 50-fold molar excess of an
unrelated YY1 oligonucleotide, or a 10-50-fold molar excess of the
mutated
BM oligonucleotide did not compete, indicating the
specificity of this binding. Nuclear extracts from undifferentiated
TNF-
-stimulated SMCs (T) formed two specific DNA-protein
complexes (B1 and B2 complexes) with
the labeled
B probe. Competition experiments demonstrated the
specificity of the two DNA-protein complexes. On the other hand, EMSA
with nuclear proteins from differentiated SMCs (D) showed no
specific DNA-protein complex. These results showed that the presence of
serum was associated with the presence of the B1 complex, TNF-
stimulation inducing the appearance of the B2 complex. These complexes
seemed to disappear in culture in defined serum-free medium, which
induced redifferentiation.

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Fig. 4.
NF- B protein binding on VCAM-1 gene B
sites is induced when SMCs are stimulated by TNF- but not when SMCs
are redifferentiated. VCAM-1 gene B sites ( B)
were used as probe in EMSAs with nuclear extracts from undifferentiated
serum-cultured SMCs (S), undifferentiated TNF- -stimulated
SMCs (T), and redifferentiated SMCs (D). Two
specific complexes (B1 and B2) can be seen.
Unlabeled competitor probe ( B), mutated B probe
( BM), and unrelated oligonucleotide (YY1) were
used for competition experiments. The x-fold molar excesses
are indicated.
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Role of NF-
B/Rel Proteins in VCAM-1 Induction on SMCs--
In
an attempt to clarify the role of NF-
B/Rel proteins in the two
conditions of VCAM-1 expression, after cytokine stimulation and during
SMC differentiation, we examined whether the antioxidant PDTC, a
blocker of NF-
B/Rel protein activation (20, 23, 35) interfered with
VCAM-1 expression on SMCs. Since it has been shown that VCAM-1 mRNA
in confluent SMCs is induced within 24 h after switching to a
defined serum-free medium (8), the experiments with PDTC were performed
at the onset of VCAM-1 mRNA induction during cytokine stimulation
or SMC differentiation. As shown in Fig.
5, as expected, TNF-
treatment alone
(lane 3) and switch to a defined serum-free medium
(lane 5) induced a strong increase in VCAM-1 mRNA
expression relative to that observed by treatment with serum
(lane 1). In the presence of PDTC, we observed a complete inhibition of the cytokine-induced VCAM-1 mRNA expression
(lane 4). A similar disappearance of the basal level of
VCAM-1 mRNA expression in SMCs maintained in serum was observed in
the presence of PDTC (lane 2). In contrast, induction of
VCAM-1 mRNA on differentiating SMCs was unaffected (lane
6). These observations indicated that the involvement of
NF-
B/Rel proteins in VCAM-1 expression depended on the status of the
SMCs.

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Fig. 5.
Antioxidant PDTC treatment prevents VCAM-1
mRNA induction in undifferentiated TNF- -stimulated SMCs but not
in redifferentiated SMCs. After pretreatment for 1 h with 100 µM PDTC, SMCs were exposed to TNF- (1000 units/ml) for
24 h or cultured during 24 h in defined serum-free medium and
in the continuous presence of 100 µM PDTC. One µg of
total RNA was used for the RT-PCR analysis. Amplified products were
electrophoresed, blotted, and hybridized with an
-32P-labeled VCAM-1 probe. Labeled DNA products were
visualized by autoradiography. Autoradiogram of PCR amplification of
retrotranscribed G3PDH mRNA is shown as control.
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|
 |
DISCUSSION |
VCAM-1 is induced or up-regulated by proinflammatory cytokines
such as TNF-
and interleukin-1
on many cellular components of the
arterial wall, including endothelial cells, SMCs, and fibroblasts (3,
39-41). More recently, VCAM-1 has been reported to be inducible by
TNF-
stimulation in a time- and dose-dependent manner on
cultured SMCs (12). The pathophysiological significance in
vivo of VCAM-1 expression on arterial SMC is unknown, although it
may amplify inflammatory processes in pathological conditions such as
atherosclerosis. However, as suggested by Rosen's work on myocytes (4)
and more recently by our own work in SMCs (8), homotypic cell-cell
interactions mediated by VCAM-1/VLA-4 may participate in the regulation
of cell differentiation. In a previous study, we demonstrated in an
in vitro model of redifferentiation, SMCs reexpressed VCAM-1 and smooth muscle-specific markers during induction of differentiation (8). These observations suggested that VCAM-1 is expressed on the
surface SMCs either after cytokine stimulation or during the process of
differentiation. These two kinds of VCAM-1 up-regulation on SMCs were
reproduced in the present experiments (cf. Fig. 1). However,
the lack of induction of sm-MHC expression after TNF-
stimulation
suggested two different mechanisms for VCAM-1 induction on SMCs.
Previous studies have shown that
B elements are involved in VCAM-1
gene induction by cytokines in endothelial cells, and for VCAM-1
expression during P19 cell differentiation into a neural pathway (6,
13). In an attempt to find out whether the VCAM-1 induction during SMC
differentiation differed from the cytokine induction pathway, we
analyzed the involvement of the NF-
B system in both processes.
Members of the NF-
B family are typically present in the cytoplasm
bound to the inhibitory I-
B proteins (21). Activation of NF-
B
involves the phosphorylation and subsequently the degradation of
inhibitory proteins (42), allowing NF-
B to translocate to the
nucleus. The members of this family, which include p50, p52, p65,
c-Rel, v-Rel, RelB, exist as homo-and heterodimers that bind to
B
sites in the enhancer regions of a large number of target genes. As in
many instances NF-
B binds as a heterodimer of a p50 in combination
with p65 to stimulate gene expression (43), we analyzed the cellular
distribution of p50 and p65 proteins in the different conditions of
VCAM-1 expression. An intense staining of the nuclear compartment was
observed for these two Rel proteins after TNF-
stimulation, the time
course of NF-
B translocation into the nucleus showing an early
nuclear staining for p50. In culture with serum, a weaker staining of
the nucleus along with a labeling of the cytoplasm was observed.
However, when the cells were cultivated for in defined serum-free
medium allowing strong VCAM-1 expression and redifferentiation process,
no immunostaining of the p50 and p65 subunits of the NF-
B/Rel family
was observed in the nuclear compartment, whereas the cytoplasm was
strongly labeled. The negative nuclear staining during the time-course experiments of NF-
B translocation into the nucleus excludes the hypothesis that NF-
B could be involved in VCAM-1 gene expression early in the differentiation stages. Cytokines such as TNF-
have been found to induce NF-
B activation in many cell types (22, 23).
Moreover, growth factors that are contained in serum such as PDGF-BB,
bFGF, EGF, and IGF-1 induce the translocation of p50 and p65 into the
nuclei of SMCs, whereas growth inhibitors such as TGF-
and
interferon-
do not induce nuclear localization of these subunits of
the NF-
B protein family (44). These findings are consistent with an
involvement of the NF-
B complex in the TNF-
-induced VCAM-1
expression we observed. The absence of p50 and p65 subunit nuclear
translocation during the redifferentiation induced by the switch to a
defined serum-free medium indicated that VCAM-1 expression was
independent of NF-
B complex activation in this case.
To directly address whether VCAM-1 expression in SMCs in different
states of differentiation was associated with NF-
B activation and
nuclear translocation, the binding activity of the
B sequence from
the human VCAM-1 promoter for the different nuclear extracts was tested
by EMSA. Specific DNA-protein complexes were observed on EMSA with
nuclear extracts of TNF-
-treated and serum-treated SMCs. Only one
DNA-protein complex, B1, was observed with serum-treated cells, and one
faster migrating, additional complex, B2, with TNF-
-treated SMCs.
Similar patterns have been reported by other workers: two NF-
B
DNA-protein complexes in phorbol ester-activated Jurkat cells; the more
rapidly migrating complex (B2) was composed of a p50 subunit and
partial degradation products of p50, while the more slowly migrating
complex (B1) contained not only these smaller proteins but
also p65 and c-Rel (45). These two complexes were also observed in the
same migration positions with nuclear extracts from rat aortic SMCs
stimulated by various growth factors (44). In our experiments, complex
B2 appeared only in human TNF-
-activated SMCs and may be associated
with VCAM-1 induction. Complex B1, described after serum stimulation
(44), did not appear to be in sufficient amount for VCAM-1 induction.
In contrast, EMSA of nuclear extracts from differentiated SMCs,
cultured for 6 days in defined serum-free medium, did not evidence any
specific DNA-protein complex. Interestingly, Lehtinen et al.
(46) reported that, after 48 h in low serum differentiation
medium, the binding activity of NF-
B was very low in all myogenic
cell lines tested (mouse C2C12 myocytes and rat L6 myocytes) and
coincided with a most abondant nuclear staining of myogenin, a protein
involved in terminal differentiation of skeletal muscle cells. In rat
aortic smooth muscle cells (between third and sixth passage) starved with 0.1% fetal bovine serum for 3 days, only a faint B1 band was
observed on EMSA by Obata et al. (44). In our experiments, the absence of the B1 band could have been due to the human origin of
the cells or to the more differentiated state of the SMCs (8). The
disappearance of a specific protein interacting with VCAM-1 gene
promoter
B sites in nuclear extracts of serum-free medium cultured
SMCs expressing VCAM-1, indicated that the NF-
B system was not
involved in its regulation during the process of differentiation.
In order to verify this idea, we analyzed the level of VCAM-1 mRNA
expression after inhibition of NF-
B system activation by an
antioxidant, PDTC. It has been shown that the activity of NF-
B as
transcriptional factor is regulated by changes in the redox state of
the cell (47). In human vascular endothelial cells, TNF-
- and
lipopolysaccharide-mediated transcriptional activation of the human
VCAM-1 promoter has been found to be mediated by NF-
B DNA elements,
and the associated NF-
B DNA-binding protein is blocked by PDTC (35).
PDTC has been shown to inhibit NF-
B stimulation by all inducers
known including H2O2 (24), by preventing dissociation of the NF-
B/I-
B complex (48). The antioxidative effect of thiol compound such as PDTC might hinge on a metal-chelating action as chelation of free iron, and copper is thought to be an
important protective mechanism against oxidants. On the other hand,
dithiocarbamates can act directly as free radical scavengers. In our
experiments, the ability of the antioxidant PDTC to prevent the
TNF-
-induced increase in VCAM-1 mRNA in SMCs supports an NF-
B-dependent signaling pathway for the
cytokine-induced VCAM-1 expression. In contrast, the persistence of
VCAM-1 mRNA expression after PDTC treatment of redifferentiated
SMCs points to an NF-
B-independent pathway in this population of cells.
Taken together, these results indicate that VCAM-1 expression on SMCs
is regulated by two independent pathways, an
NF-
B-dependent pathway after cytokine activation and an
NF-
B-independent pathway during the redifferentiation process.
Several mechanisms could account for the regulation of VCAM-1
expression during the redifferentiation process, either nonspecific or
specific. Among the nonspecific mechanisms of VCAM-1 regulation during
differentiation, an autocrine/paracrine activation comes to mind.
However, the lack of involvement of NF-
B proteins in the
transcriptional regulation suggests that VCAM-1 expression is
independent of an autocrine/paracrine stimulation by growth factors
such as PDGF-BB, bFGF, EGF, and IGF-1 or cytokines as TNF-
and
interleukin-1
synthesized by arterial wall cells. All these secreted
factors are known to activate the NF-
B system. Another possibility
is the involvement of pleiotropic transcription factors, different from
the NF-
B system. Two GATA DNA elements are present in opposite
transcriptional orientations in the VCAM-1 gene promoter at positions
between
255 and
235 relative to the transcription start site. GATA
zinc finger proteins, which are involved in TNF-
induction of VCAM-1
in endothelial cells, belong to a transcription factor family composed
of at least eight members. GATA-1, -2, and -3 are involved in
differentiation of hematopoietic cells. GATA-4 has a tissue
distribution limited to heart and endodermally derived tissue.
Furthermore, inhibition of GATA-4 protein expression blocks cardiac
muscle differentiation in vitro. Recently, GATA-6 human
transcripts have been detected in lung and liver and in cultures of
human and rat vascular SMCs (49). In SMCs, GATA-6 transcripts are
down-regulated when quiescent cultures are stimulated to proliferate in
response to mitogen activation. As withdrawal from the cell cycle is a
prerequisite for differentiation, it is possible that the GATA-6
protein participates in regulation of the differentiative state of SMCs
and thus to induction of the VCAM-1 gene expression. However, a search
for GATA DNA-binding proteins in redifferentiated SMC nuclear extracts
using the GATA probe on EMSA did not uncover any specific DNA-protein
complexes (data not shown).
VCAM-1 expression could be also regulated by a muscle-specific
differentiation pathway. During striated muscle differentiation, VCAM-1
is expressed in a developmentally specific patter. The cell-cell
interactions underlying myogenesis have been shown to involve an
interaction between VCAM-1 and VLA-4 (4). Study of VCAM-1 promoter
activity in C2C12 mouse myoblasts has identified a position-specific
enhancer located between bp
17 and
5, consensus for an interferon
regulatory factor (IRF) binding element that overrides the effect of
the other promoter elements, resulting in VCAM-1 constitutive
expression (50, 51). The transcription factor IRF-2 that interacts with
this VCAM-1 gene promoter in muscle cells is expressed concomitantly
with VCAM-1 in differentiating skeletal muscle in mouse embryos.
Similarly, VCAM-1 expression has been described in human aorta during
ontogenesis (8), suggesting that VCAM-1 could be regulated by this IRF
binding site. However, in human differentiated SMCs, we did not find
any specific protein-DNA complex by EMSA with this element (data not
shown). These negative results suggested that IRF binding site is not
involved in SMC expression of VCAM-1.
These results led us to suspect a smooth muscle-specific
transcriptional regulation of VCAM-1 gene in differentiated SMCs. Several studies on smooth muscle gene promoters (SM22
, sm-MHC, sm-
actin) have found evidence for roles of various transcription factors such as MEF2 (52, 53) and CArG-box binding proteins (53, 54).
Several potential CArG-boxes and MEF2 binding sites are present in the
VCAM-1 gene promoter. The isoforms of the MEF2 transcription factor
belonging to the MADS box family transcription factors are expressed in
vascular SMCs and in vascular tissue (55) and during
Drosophila ontogenesis, MEF2 is required for later aspects
of differentiation of the three major types of musculature (56, 57).
MEF2 binding sites are found in the control of the majority of
muscle-specific genes (58). Interestingly, a MEF2-like binding site is
present in the VCAM-1 gene promoter located from
1992 to
1982
(5'-TTTTAATAAA-3'). It resembles the MEF2-like sequence of the rabbit
sm-MHC gene promoter found at
1540 to
1530 (5'-TATTAATATAA-3')
involved in gene expression in rat aortic SMCs (52). It is of note that
this MEF2-like binding site specifically interacts with MEF2B (59).
These findings suggest that many potential smooth muscle-specific
cis-regulator elements in the VCAM-1 gene promoter could participate in
the regulation of VCAM-1 during redifferentiation of smooth muscle
cells. This might constitute a smooth muscle-specific pathway. Further
experiments will be required to gain further understanding of the
mechanisms underlying the intriguing pattern of VCAM-1 expression in
smooth muscle tissue.