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INTRODUCTION |
Syndecan-4 is one of the principal heparin sulfate-carrying
proteins on the cell surface (1). It is expressed by a number of
different cell types including endothelial cells, smooth muscle cells,
and cardiac myocytes, although expression in quiescent tissues is
fairly low. Syndecans can participate in a variety of biological
functions including regulation of blood coagulation, cell adhesion, and
low density lipoprotein transport (2, 3). In addition to these
activities shared by all syndecans, syndecan-4 cytoplasmic domain can
bind to and activate protein kinase C-
with the degree of activation
regulated by the extent of syndecan-4 cytoplasmic tail phosphorylation
(4-7).
Despite these interesting properties, little is known about the
regulation of syndecan-4 gene expression. Its levels are increased after various forms of tissue injury including skin wounds (8), vascular wall injury (9), or myocardial infarction (10). The increase
in syndecan-4 expression in skin wounds and in the heart following
myocardial infarction has been ascribed to the presence of a
proline-arginine-rich peptide, PR39. No other factors responsible for
this increase have been identified (8, 10). The present study was
designed to study the role of cardiac myocytes in the induction of
syndecan-4 expression under normal and ischemic (hypoxic) conditions.
To this end, we determined the ability of myocyte- or
myoblast-conditioned medium to induce syndecan-4 expression in a human
endothelial cell line. We find that
TNF-
1 secreted by hypoxic
but not normal myocytes is able to induce syndecan-4 expression and
that the mechanism of this induction involves both transcriptional
activation of syndecan-4 gene expression and posttranscriptional
regulation of syndecan-4 message half-life.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
A human umbilical endothelial cell line,
ECV304 (ATCC), was cultured in M199 medium supplemented with 10% fetal
bovine serum (FBS), 100 units/ml penicillin, and 100 µg/ml
streptomycin. A rat cardiac myoblast cell line, H9c2 (ATCC), was
cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% FBS and penicillin/streptomycin. IC-21 (ATCC), a mouse
macrophage cell line, was cultured in RPMI 1640 medium supplemented
with 10% FBS.
Cardiac myocytes were isolated from day 2 neonatal C57/129 or same
background TNF-
/
mice (11). A total of 24 hearts
from each mouse strain was used. At sacrifice the hearts were removed
aseptically, cut into 1-2-mm pieces, placed in ice-cold Hanks'
balanced saline solution without either Ca2+ or
Mg2+, and subjected to serial digestions with 0.5 mg/ml
collagenase II (Worthington), 0.1% trypsin, 15 µg/ml DNase I, and
1% chicken serum at 37 °C with constant rotation. Supernatant was
collected every 10-15 min, and digestion was stopped by addition of
10% horse serum. Dissociated cells were collected by centrifugation and resuspended in DMEM/F12 medium supplemented with 5% horse serum, 2 g/liter bovine serum albumin (fraction V), 3 mM pyruvic acid, 15 mM HEPES (pH 7.6), 100 µM ascorbic
acid, 100 units/ml penicillin, 100 µg/ml streptomycin, 4 µg/ml
transferrin, 0.7 ng/ml sodium selenium, and 5 µg/ml linoleic acid for
1 h to enrich for myocytes. The suspension was then plated onto
1% gelatin-coated dishes at 2.5 × 106 cells/60-mm
dish. Bromodeoxyuridine (0.1 mM) was added to prevent proliferation of non-myocytes. Horse serum and bromodeoxyuridine were
removed from the medium after 36 h.
Growth Factor Studies--
For determination of growth factor
responsiveness of syndecan-4 expression, ECV304 cells were cultured in
10% FBS-DMEM to 80% confluence. At that point the cells were washed
with PBS, the medium was replaced with serum-free DMEM for 6 h,
and then one of the following growth factors was added:
hrVEGF165 (25 ng/ml, Genentech, Inc.), hrbFGF (25 ng/ml,
Chiron Corp.), hrPDGF-AA (20 ng/ml, Sigma) or hrPDGF-AB (20 ng/ml,
Sigma), hrTGF-
(20 ng/ml, R&D Systems), hrTNF-
(20 ng/ml, Sigma),
or mouse epidermal growth factor (20 ng/ml, Becton Dickinson Labware).
Cells were harvested and total RNA was extracted after 24 h and
subjected to Northern blot analysis.
Preparation of Myoblast- or Myocyte-conditioned Medium--
H9c2
cells were cultured in DMEM supplemented with 10% FBS in 100-mm dishes
to 90% confluence. At that point, the cells were washed three times
with PBS and the medium was changed to serum-free DMEM. Following
24 h of culture under hypoxic (0-2% O2) or normal oxygen conditions, the medium was collected, centrifuged for 10 min at
2000 × g to remove debris, and then stored at
80 °C for further use. Similar procedures were used for collection
of myocyte-conditioned medium.
Analysis of Syndecan-4 Expression--
For Northern analysis,
total RNA extracted from cultured cells with Tri Reagent (Sigma) was
fractionated on 1.2% formaldehyde-agarose gel and transferred onto
GeneScreen Plus membrane. cDNA probes for human syndecan-4 or
glyceraldehyde-3-phosphate dehydrogenase were labeled with
-[32P]dCTP using a random priming kit (Roche Molecular
Biochemicals), and hybridization was carried out for 3 h at
65 °C in Quickhyb solution (Strategene), followed by serial washes
with 0.1× SSC, 0.1% SDS at room temperature for 15 min, 45 °C for
15 min, and 65 °C for 15 min. The quantitative analysis of
hybridization signal was carried out using PhosphorImager (Molecular
Dynamics) and ImageQuant software. RNA loading was adjusted using
ethidium bromide gel 18 S RNA signal or glyceraldehyde-3-phosphate
dehydrogenase expression level.
For determination of syndecan-4 protein levels, ECV cells were washed
with PBS (137 mM NaCl, 10 mM
Na2HPO4, 3.6 mM KCl, 1.8 mM KH2PO4 (pH 7.4)), dissociated by
0.05% trypsin, 0.5 mM EDTA (Life Technologies, Inc.) in
PBS for 10 min at 37 °C, and sedimented by 200 × g
centrifugation at 4 °C for 5 min. Western blotting was carried out
with anti-syndecan-4 cytoplasmic domain antibody following
SDS-polyacrylamide gel electrophoresis of the total cell protein
extract as described previously (6).
Western Blot Analysis of TNF-
in Myoblast-conditioned
Medium--
Conditioned medium collected from five 100-mm Petri dishes
of confluent H9c2 cells cultured for 48 h under hypoxic or normal conditions was concentrated using Centriprep followed by Centricon columns (Amicon, Inc.) to a final volume of 80 µl. Aliquots of the
concentrated material (20 µl) were subjected to 10%
SDS-polyacrylamide gel electrophoresis and transferred to an
ImmobilonTM-P membrane (Millipore), and the blot was then
incubated with a polyclonal goat anti-rat TNF-
antibody (1:200
dilution, Santa Cruz Biotechnology) in PBS supplemented with 5% nonfat
milk overnight at 4 °C. An ECL system (Amersham Pharmacia Biotech)
was used to detect the signal.
Syndecan-4 mRNA Stability Assay--
100 µM
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole was added
to ECV304 cells following overnight culture in 0.5% FBS-M199. The
cells were then harvested at the indicated time points and total RNA
was extracted as described above. 10 µg of total RNA for each time
point were subjected to Northern blot analysis with a syndecan-4
cDNA probe. Signal was quantified using PhosphorImager with RNA
loading adjusted by glyceraldehyde-3-phosphate dehydrogenase levels.
The mRNA half-life was calculated using the formula
t1/2 = ln2/(mRNA decay rate constant) with the
decay rate constant derived from the slope of the decay curve.
Syndecan-4 Transcription Studies--
A 715-nucleotide fragment
(
690 to +25 nucleotide of the murine sequence) encompassing the basal
elements of mouse syndecan-4 promoter (courtesy of Dr. T. Kojima,
Nagoya University) was cloned into a pGL-3 vector (Promega) containing
the luciferase reporter construct. The syndecan-4 promoter construct
and a pGRE-Neo (U. S. Biochemical Corp.) construct were linearized
and, following phenol/chloroform purification, used for
electroporation. To this end, ECV304 cells were trypsinized and washed
twice with a fresh medium; 6 × 106 of cells in 0.75 ml of medium were incubated with 30 µg of syndecan-4 promoter-containing pGL-3 and 3 µg of pGRE-Neo linearized plasmids for 5 min on ice in a 0.4-cm cuvette. Electroporation was carried out
at 290 V, 600 microfarads using Gene Pulser II (Bio-Rad). Following
electroporation, the cells were quickly diluted with the fresh M199
medium containing 10% FBS supplemented with 5 mM sodium
butyrate and plated onto a 100-mm Petri dish. Sodium butyrate was
removed after overnight exposure, and the culture was continued for
another 36 h in 10% FBS-M199. The cells were then passaged into
two dishes and cultured in medium containing 1 mg/ml G418 (Life
Technologies, Inc.). All colonies were pooled together after 10 days
and maintained in the selection medium.
For promoter studies, cells stably expressing syndecan-4 promoter
construct were plated onto 12-well plates and allowed to come to 80%
confluence in 10% FBS-M199 medium. The medium was then changed to
0.5% FBS-M199 overnight. TNF-
was added at the indicated
concentration. After 6 h of exposure, cells were lysed, and
luciferase activity was determined using the luciferase assay system (Promega).
To assess the role of NF-
B in the TNF-
-dependent
stimulation of syndecan-4 gene expression, ECV304 cells plated on 60-mm dishes were cultured until 90% confluence and subjected to serum starvation overnight. Lactacystin (Calbiochem) was then added to medium
at a concentration of 10 µM. 1 h later, TNF-
(20 ng/ml) was added. Syndecan-4 mRNA levels were determined using
Northern blot analysis 4 h after exposure of lactacystin-treated
or control cells to TNF-
(20 ng/ml).
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RESULTS |
Induction of Syndecan-4 Expression in Endothelial Cells by Hypoxic
Myoblast Cell-conditioned Medium--
To investigate the role of
myocyte-endothelial cell interaction in the regulation of syndecan-4
expression in endothelial cells, we studied the ability of myoblast- or
cardiac myocyte-conditioned medium to induce syndecan-4 expression in
ECV304 cells. Exposure of ECV304 cells to the medium conditioned by the
H9c2 cells cultured under normal conditions did not affect syndecan-4
expression. However, medium conditioned by H9c2 cells cultured under
hypoxic conditions produced a nearly 2-fold increase in syndecan-4
mRNA expression in ECV304 cells and a 2.5-fold increase in syndecan protein (Fig. 1). These results suggested
that myoblasts exposed to hypoxia secreted a factor that was
responsible for induction of syndecan-4 expression. To determine
whether any of the known myoblast-secreted growth factors were
responsible for this event, ECV304 cells were exposed to a panel of
cytokines. Of the growth factors tested, only TNF-
was able to
induce syndecan-4 expression in ECV304 cells (Fig.
2) in a dose-dependent
manner. The induction of syndecan-4 mRNA expression by TNF-
was
prompt, beginning at 1 h, reaching maximal effect at 3 h
(Fig. 2), and lasting for at least 24 h (data not shown).

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Fig. 1.
Effects of H9c2-conditioned medium on
syndecan-4 expression in ECV304 cells. Syndecan-4 gene expression
in ECV304 cells was assessed after 24 h of exposure to the medium
conditioned by hypoxic (lane 2) or normal (lane
3) H9c2 myoblasts using Northern (A) or Western
(B) blotting. Note a significant induction in syndecan-4
mRNA and protein expression by medium conditioned by hypoxic but
not normal H9c2 cells. C, graphic representation of the
effects of H9c2-conditioned medium on syndecan-4 mRNA and protein
expression. Syndecan-4 mRNA levels are expressed as percent control
(mean of two determinations). Black bars, mRNA levels;
striped bars, protein levels; H9c2-CM, H9c2
myocyte-conditioned medium.
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Fig. 2.
Role of TNF- in
induction of syndecan-4 expression. A, growth factor
induction of syndecan-4 expression. Subconfluent serum-starved ECV304
cells were treated with either vascular endothelial growth factor
(VEGF, 25 ng/ml), basic fibroblast growth factor
(bFGF, 25 ng/ml), platelet-derived growth factor-AA
(PDGF-AA, 20 ng/ml), platelet-derived growth factor-AB
(PDGF-AB, 20 ng/ml), transforming growth factor-
(TGF- , 20 ng/ml), TNF- (20 ng/ml), or epidermal growth
factor (EGF) (20 ng/ml). Total RNA was harvested after
24 h and subjected to Northern analysis for syndecan-4 expression.
B and D, time course of TNF- -mediated
induction of syndecan-4 expression. Subconfluent serum-starved ECV304
cells were treated with 20 ng/ml TNF- and subjected to Northern
(B) or Western (D) blotting with anti-syndecan-4
cytoplasmic domain antibody at indicated time points. C and
E, dose response of TNF- -dependent induction
of syndecan-4 expression. Indicated amounts of recombinant TNF- were
added to subconfluent ECV304 cells, and syndecan-4 mRNA
(C) and protein levels (E) were determined 6 h later. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
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TNF-
Is a Primary Factor Responsible for Induction of Syndecan-4
Expression by Myoblasts--
To demonstrate that TNF-
is produced
by hypoxic myoblasts, cell culture medium conditioned by H9c2 cells
under normal and hypoxic conditions was subjected to Western analysis
with anti-TNF-
antibody. Cell lysate of IC-21 cells, a macrophage
cell line known to produce large amounts of TNF-
under normal
culture conditions, was used as a control. The presence of secreted
TNF-
was detected in IC-21 cell lysate and in H9c2 cells cultured
under hypoxic conditions. Compared with the hypoxic H9c2
cell-conditioned medium, only a trace amount of TNF-
was detected in
the medium conditioned by the normal H9c2 cells (Fig.
3A). To confirm that TNF-
is the primary factor responsible for myoblast-dependent
induction of syndecan-4 expression, we used primary myocyte cultures
from homozygous TNF-
knockout mice and age-matched controls to
condition serum-free medium in the manner similar to the H9c2
experiments. Cell culture media conditioned by primary myocytes derived
from either the TNF-
/
or wild type C57/129 mice
cultured under normal conditions did not induce endothelial cell
syndecan-4 mRNA expression. However, medium conditioned by hypoxic
myocytes from the C57/129 but not TNF-
/
mice induced
syndecan-4 expression to the extent similar to that seen with hypoxic
H9c2 cells (Fig. 3B).

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Fig. 3.
TNF- produced by
ischemic myocytes is the primary factor responsible for induction of
syndecan-4 expression. A, Western blot analysis of
secreted TNF- protein in medium conditioned by H9c2 cells cultured
under normal or hypoxic conditions. Note the increased presence of
TNF- in the medium conditioned by hypoxic (lane 3) but
not normal (lane 2) H9c2 cells. IC-21 cell lysate
(lane 1) was used as a control. B, to demonstrate
the dominant role of TNF- in induction of syndecan-4 expression,
primary myocytes isolated from neonatal hearts of TNF- knockout
(TNF- / ) and age-matched control
(TNF- +/+) mice were used to condition medium
during culture under normal or hypoxic conditions. Northern analysis of
syndecan-4 expression in ECV304 cells 24 h after exposure to
conditioned medium demonstrated increased syndecan-4 levels only in
cells exposed to the medium conditioned by hypoxic myocytes from
control mice (B, top). A graphic representation
of syndecan-4 mRNA expression level after normalization for 18 S
RNA ribosomal band is shown (B, bottom). All
results are expressed as percent control (syndecan-4 levels in ECV
cells prior to any exposure).
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Mechanism of TNF-
-mediated Induction of Syndecan-4
Expression--
To determine whether TNF-
regulates syndecan-4 gene
expression by transcriptional or posttranscriptional mechanisms, we
measured syndecan-4 mRNA half-life in ECV304 cells in the presence
or absence of TNF-
treatment. Exposure to TNF-
at 20 ng/ml
increased syndecan-4 mRNA half-life from 16 to 24 h (Fig.
4A). To assess the effect of
TNF-
on syndecan-4 gene transcription, we measured activity of a
luciferase construct under control of the mouse syndecan-4 promoter.
Exposure of the pooled population of ECV304 cells stably transfected
with this construct to TNF-
at 20 ng/ml led to a 2-fold increase in
luciferase activity (Fig. 4B). Because TNF-
is known to
activate gene expression in an NF-
B-dependent manner and given that syndecan-4 promoter contains an NF-
B-binding site, we
examined the effect of inhibition of NF-
B-dependent
transcription on the ability of TNF-
to induce syndecan-4
expression. Pretreatment with lactacystin, an inhibitor of
proteasome-dependent degradation of I
B
expression,
completely blocked TNF-
-induced activation of syndecan-4 expression
(Fig. 5). These results indicate that TNF-
regulates syndecan-4 expression at both transcriptional and
posttranscriptional levels.

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Fig. 4.
Mechanism of
TNF- -induced increase in syndecan-4 expression
in ECV cells. A, to assess the effect of TNF-
administration on syndecan-4 mRNA half-life, ECV304 cells were
cultured with or without TNF- (20 ng/ml). Following overnight
exposure, 5,6-dichloro-1- -D-ribofuranosylbenzimidazole,
at a concentration of 100 µM, was added. At indicated
time points, the cells were harvested and subjected to Northern blot
analysis for syndecan-4 expression. Signal was quantified using
PhosphorImager with RNA loading adjusted for glyceraldehyde-3-phosphate
dehydrogenase levels. Note the substantially prolonged syndecan-4
mRNA half-life in TNF- -treated cells. B, to assess
the effects of TNF- administration on syndecan-4 promoter activity
in ECV304 cells, we conducted an analysis of luciferase activity in
pools of ECV cells stably transfected with a construct containing mouse
syndecan-4 basic promoter fragment ( 690 to +25 nucleotide) linked to
a luciferase gene. Note a 2-fold increase in luciferase activity
following TNF- treatment (mean ± standard deviation,
p < 0.01 TNF- treatment versus control).
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 5.
TNF- induces
syndecan-4 gene expression in an
NF- B-dependent manner. To
study the role of NF- B-dependent gene transcription in
TNF- -induced activation of syndecan-4 expression, ECV304 cells were
exposed to TNF- (20 ng/ml) in the absence of lactacystin
(TNF- ) or following 1 h pretreatment with
lactacystin (LC+TNF- ). Note the increased syndecan-4
expression in TNF- -treated cells that were blocked by pretreatment
with lactacystin. Addition of lactacystin by itself (LC) had
no effect on base-line syndecan-4 levels. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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DISCUSSION |
The principal finding of this study is that TNF-
, secreted by
hypoxic but not normal myocytes, is able to increase syndecan-4 expression in endothelial cells. A number of findings point to TNF-
as the main factor responsible for the ability of hypoxic myocyte-conditioned medium to induce syndecan-4 expression. First, the
cytokine is capable of increasing syndecan-4 mRNA levels in a
dose-dependent manner and with an appropriate time course.
Second, TNF-
is present in medium conditioned by hypoxic but not
normal myoblasts. Finally, medium conditioned by myocytes cultured
under either normal or hypoxic conditions derived from the TNF-
knockout mice fails to induce syndecan-4 expression.
TNF-
-dependent activation of syndecan-4 expression
involves both transcriptional and posttranscriptional events. The study of syndecan-4 transcription was carried out using a mixture of approximately 200 independent stable clones, thus eliminating artifacts
potentially associated with the expression construct integration into
hyper- or hypoactive regions of the genome. The promoter fragment used
was previously shown to possess basal promoter activity (12), although
it is possible that additional control elements were missing. The
degree of induction of the luciferase expression was similar to the
extent of increase in the endogenous syndecan-4 mRNA level in
TNF-
-treated ECV304 cells. Taken together, the extent of
transcriptional activation as measured in luciferase assays and
prolongation of mRNA half-life closely parallel the increase in
endogenous syndecan-4 protein levels in these cells following TNF-
exposure.
TNF-
is thought to regulate gene expression by inducing degradation
of a transcriptional inhibitor, I
B
, thus allowing the p50
NF-
B·RelA complex to bind to its nuclear binding sites and activate gene expression (13). Basal syndecan-4 promoter possesses an
NF-
B binding site (GGGGGAATT, nucleotides
84 to
76 of the mouse
sequence). Therefore, it is possible that syndecan-4 gene expression
up-regulation by TNF-
occurs, at least in part, via the
NF-
B-dependent pathway. To confirm this, we exposed
ECV304 cells to TNF-
following pretreatment with lactacystin.
Lactacystin is a specific inhibitor of proteasome function (14) and, in particular, is known to block proteasome-dependent
degradation of I
B
. In accordance with these considerations, we
found that pretreatment with lactacystin completely blocked the ability
of TNF-
to induce syndecan-4 expression.
Recent studies have suggested that syndecan-4 plays an important role
in regulation of endothelial cell growth and migration and in basic
fibroblast growth factor-dependent signaling (4, 5, 7).
Studies in our laboratory have suggested that increased syndecan-4
expression augmented the ability of endothelial cells to migrate,
proliferate, and form vascular structures in Matrigel in response to
basic fibroblast growth
factor.2 Thus, the ability of
TNF-
to induce syndecan-4 expression while suppressing that of
syndecan-1 (15) may lead to enhanced endothelial cell growth.
TNF-
is a potent angiogenic factor in a number of in
vitro and in vivo systems (16, 17). The cytokine
expression in the heart is induced by myocardial ischemia (18, 19), and
the presence of secreted TNF-
has been demonstrated in an in
vitro model (20). Whereas TNF-
has a wide range of biological
activities, its ability to induce syndecan-4 expression may play a
particularly important role in mediation of its angiogenic activity.
In summary, ischemic myocytes can induce syndecan-4 expression in
surrounding endothelial cells by paracrine secretion of TNF-
. This
mechanism may play an important role in myocyte-dependent regulation of endothelial cell activity and the ability of ischemic tissues to initiate and propagate an angiogenic response.