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From the Centre for Thrombosis and Vascular Research, School of Pathology, The University of New South Wales, and Department of Haematology, Prince of Wales Hospital, Sydney, Australia
| Abstract |
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| Introduction |
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Deliberate denudation of endothelium in rat arteries without traumatizing underlying smooth muscle cells triggers chemotaxis and proliferation at the site of injury.7 The first smooth muscle cells to migrate from the media to the intima in this model do so preferentially at the endothelial wound edge,8 suggesting that factors produced by injured endothelium can influence this process. Factors such as fibroblast growth factor-2 (FGF-2) are basally expressed in endothelial cells and smooth muscle cells in the artery wall. The inappropriate release of FGF-2 and/or other endogenous factors during catheterization or other forms of injury may initiate molecular events that lead to lesion formation.
The promoters of many genes whose products can influence cell movement and replication in the vessel wall, such as FGF-2,9PDGF-A,911PDGF-B,10,12 and TGF-ß19,13 bear nucleotide recognition elements for the zinc finger transcription factor, early growth response factor-1 (Egr-1).14-17 Egr-1 (also known as TIS8, krox-24, and NGFI-A)18 is an immediate-early gene product that binds preferentially to GC-rich motifs in DNA.19,20 Egr-1 is rapidly induced at the endothelial wound edge before the increased expression of growth factors at the same location.14 Here, we define signaling pathways triggered by endothelial injury and growth factor release that lead to the paracrine activation of this pleiotropic transactivator.
| Materials and Methods |
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Bovine aortic endothelial cells (Cell Applications) were grown in Dulbecco's modified Eagles' medium (DMEM; Life Technologies), pH 7.4, containing 10% fetal bovine serum (FBS), 10 U/ml penicillin, and 10 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. All cells in the experiments were used between passages 1 and 7. Cells were rendered quiescent by incubation in DMEM, pH 7.4, containing 0.25% plasma-derived serum (PDS) for 24 hours. The cells were injured by scraping with a sterile stainless steel comb or exposed to the agonists indicated. Lysates were prepared by the addition of 10 mmol/L Tris/HCl, 150 mmol/L NaCl, 2 mmol/L EGTA, 2 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin, pH 7.4, at 4°C, and protein concentrations were assessed using the Biorad protein assay. ERK1/2 activity was assessed using the p44/p42 MAP kinase system (Amersham) in accordance with manufacturer's instructions.
Western Blot Analysis
Lysates were resolved by electrophoresis on denaturing 10% SDS-polyacrylamide gels for 2 hours at 100 V. Proteins were transferred to nylon membranes, nonspecific binding sites were blocked with nonfat skim milk, and the membranes were incubated with polyclonal antibodies raised against Egr-1 (1:250; Santa Cruz) or monoclonal antipeptide antibodies recognizing phosphorylated forms of JNK-1 and JNK-2 (1:250; Santa Cruz) before chemiluminescent detection (NEN-DuPont). Where indicated, the Egr-1 blot was stripped and reprobed with antibodies to Sp1 (1:250; Santa Cruz).
FGF-2 ELISA
FGF-2 release into the supernatant was assessed using a commercial
ELISA specific for this growth factor (R&D Systems). This kit does not,
according to the manufacturer, recognize FGF-1, FGF-4, FGF-5, FGF-6,
keratinocyte growth factor, or ß-endothelial cell growth factor. It
also fails to recognize interleukin (IL)-1, IL-2, IL-3, IL-4, IL-6,
IL-7, IL-8, epidermal growth factor (EGF), granulocyte
colony-stimulating factor, granulocyte/macrophage colony-stimulating
factor, HB-EGF, hepatocyte growth factor, insulin-like growth
factor (IGF)-I, IGF-II, LIF, ß-nerve growth factor,
PD-endothelial cell growth factor, transforming growth factor
(TGF)-
, TGF-ß1, tumor necrosis factor (TNF)-
,
TNF-ß, vascular endothelial growth factor, or platelet-derived growth
factor (PDGF)-AB. Values were normalized to the concentration of
protein in the supernatant.
Electrophoretic Mobility Shift Analysis
Binding reactions were performed with approximately 10 µg of nuclear extract21 in 20 µl containing 1 µg of poly(dI/dC)-poly(dI/dC) (Sigma), 1 µg of salmon sperm DNA (Sigma), 10 mmol/L Tris/HCl, pH 7.5, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 5% glycerol, and the 32P-labeled egr-1SRE (120,000 cpm) for 35 minutes at 22°C. In competition or supershift studies, a 150-fold molar excess of unlabeled oligonucleotide or 2 µg of affinity-purified rabbit anti-peptide antibody (Santa Cruz) was included in the binding mixture 10 minutes before addition of the probe. Bound complexes were separated from the unbound probe by nondenaturing 6% polyacrylamide gel electrophoresis and 1X Tris-buffered borate/EDTA running buffer at 200 V. Gels were dried and exposed to Hyperfilm-MP (Amersham) overnight at -80°C.
Transient Transfection and Reporter Gene Analysis
Endothelial cells were transiently transfected with 10 µg of egr-1 promoter-chloramphenicol acetyl transferase (CAT) reporter construct using Superfect (Qiagen). FGF-2 (25 ng/ml) (final endotoxin concentration less than 1 pg/ml) was incubated with growth-quiescent cells for 24 hours. CAT activity was assessed as previously described22 and normalized to the concentration of protein in the cell lysates.
| Results and Discussion |
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Paracrine Activation of Egr-1 by FGF-2
FGF-2 lacks a conventional hydrophobic sequence for exocytotic
secretion.26
Consequently, it is not efficiently
transported outside endothelial cells and is instead localized mainly
within the cytoplasm and nucleus in vitro and in
vivo.7,27,28
To preempt a role for endogenous FGF-2 in
the induction of Egr-1, we loaded endothelial monolayers with
51Cr before injury by scraping. Maximal release of
radiolabel was observed within seconds of the mechanical insult (Figure 1A)
. To provide direct evidence for the
release of FGF-2, we performed a solid-phase ELISA specific for FGF-2
(Figure 1B)
. FGF-2 antigenic activity was readily detected in the
supernatant within 2 minutes of scraping (Figure 1B)
.
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An electrophoretic mobility shift analysis (EMSA) was performed using
nuclear extracts of injured cells with a 32P-labeled
double-stranded oligonucleotide (32P-Oligo A) bearing the
proximal region (bp -76/-47) of the PDGF-A promoter.30
This region contains overlapping consensus Egr-1 binding
sites22
and mediates inducible PDGF-A expression in
response to multiple agonists of Egr-1.21,22,31
A distinct
nucleoprotein complex was observed when extracts from injured, but not
uninjured, cells were used in the EMSA (Figure 1D)
. Formation of this
inducible complex was strongly inhibited by incubation of the cells
with FGF-2 antibodies, but not with nonspecific IgG, before injury
(Figure 1D)
. The complex was completely supershifted with antibodies to
Egr-1 (Figure 1D)
. These data, taken together, indicate that Egr-1
activation, its nuclear translocation, and interaction with the PDGF-A
promoter in endothelial cells after injury is contingent on the local
release of FGF-2.
SRF Binds egr-1 Promoter in Endothelial Cells
Inducible Egr-1 transcription is dependent on cooperative
interactions between serum response factor (SRF, p67)32
and
ternary complex factors (TCFs)33
at serum response elements
(SREs) in the egr-1 promoter.34-40
SRF binds as
dimers to the central CArG box of the SRE, whereas TCFs (such as Elk-1,
SAP-1a, and SAP-2a) are activated by phosphorylation and interact
directly with SRFs and/or DNA as monomers or dimers.41
The
egr-1SRE located at nucleotides -374/-355 mediates
inducible promoter-dependent expression in response to multiple
agonists and stresses.35,42,43
An EMSA was performed using
a 32P-labeled oligonucleotide bearing the -374/-355
region of the egr-1 promoter. This resulted in the formation
of two distinct nucleoprotein complexes (Figure 2A)
. A 150-fold molar excess of either
unlabeled egr-1SRE or an oligonucleotide bearing the
c-fosSRE that contains recognition elements for Elk-1 and
SRF, abrogated formation of these complexes (Figure 2A)
. In contrast,
an irrelevant oligonucleotide, P-mSSRE, had no inhibitory effect
(Figure 2A)
. Both complexes were supershifted (complex S) by antibodies
recognizing SRF (Figure 2A)
, whereas neither complex was affected by
antibodies directed against transcription factors PEA-3 (Figure 2A)
,
Egr-1 (Figure 2A)
, WT-1, or AP2 (data not shown). These findings
demonstrate hitherto unreported SRF binding activity in endothelial
cells. Surprisingly, positive supershifts were not observed using
antibodies directed toward native or phosphorylated Elk-1 (at Ser-383),
either before or after exposure to FGF-2 (Figure 2A
and data not
shown).
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Although the precise TCF(s) mediating inducible egr-1
transcription is not known, all members of this Ets family of
trans-acting factors are activated by carboxyl-terminal
phosphorylation by extracellular-signal-regulated kinase (ERK), the
downstream target of MEK. Transient transfection analysis in
endothelial cells using a CAT reporter vector driven by a fragment of
the egr-1 promoter bearing the -374/-355 SRE and four
additional SREs downstream18
determined that
egr-1-promoter-dependent CAT activity was induced on
exposure to FGF-2 (Figure 2B)
. PD98059, a flavone that binds inactive
MEK and prevents phosphorylation by Raf without affecting other known
serine/threonine or tyrosine kinases44
abrogated FGF-2
activation of the egr-1 promoter. FGF-2-inducible
egr-1-promoter-dependent expression was also abolished by
the phosphatidylinositol 3-kinase inhibitor, wortmannin (Figure 2B)
.
Inhibition by PD98059 and wortmannin was dose dependent and maximal at
20 µmol/L and 1 µmol/L, respectively (Figure 2B
and data not
shown).
Co-transfection experiments further linked ERK signaling with the
egr-1 promoter in endothelial cells. CAT activity inducible
by FGF-2 was blocked by overexpression of dominant negative mutants of
ERK-1/2 (Figure 2B)
. Overexpression of the vector alone was without
effect (Figure 2B)
. Cells transfected with the backbone CAT plasmid or
a PDGF-A promoter-CAT reporter construct, f36, which bears 55 bp of
PDGF-A promoter sequence and has its 5' endpoint located 3' to the
Egr-1 binding site,22
showed no increase in CAT activity in
cells incubated with FGF-2 (data not shown). Moreover, neither
co-expression of the ERK-1/2 dominant negative construct nor exposure
to inhibitors had any effect on reporter activity (data not shown).
Endothelial Injury Activates ERK
To provide direct evidence for the activation of ERK as a
consequence of endothelial injury, we performed a
[
32P]ATP incorporation assay using a peptide substrate
specific for this mitogen-activated protein kinase. ERK-1/2 enzymatic
activity increased fivefold within 2 minutes of injury (Figure 3)
. Recombinant FGF-2 also stimulated
ERK-1/2, whereas TNF-
had no measurable effect (Figure 3)
.
Monolayers were incubated with FGF-2 antibodies before injury to
determine whether ERK-1/2 activity after scraping was dependent on
cellular FGF-2. These antibodies suppressed ERK-1/2 activity 8 minutes
after injury (Figure 3)
. Thus, ERK and Egr-1 activation after injury is
mediated in part by the release and paracrine action of endogenous
FGF-2.
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TCFs can also be activated by members of the JNK family of
mitogen-activated protein kinases.45-48
Wortmannin has
been reported to inhibit JNK activity without affecting
ERK.49-52
As wortmannin blocked FGF-2 activation of the
egr-1 promoter (Figure 2B)
, we determined whether JNK was
itself phosphorylated by injury. Using monoclonal antibodies
recognizing phospho-Thr-183 and Tyr-185 in JNK-1 and JNK-2, we observed
time-dependent JNK-1 activation by injury but could not detect
phosphorylated JNK-2 (Figure 4)
. JNK-1
phosphorylation after 8 minutes was inhibited by preincubation of the
monolayers with FGF-2 antibodies (Figure 4)
. Recombinant FGF-2, like
injury, strongly stimulated JNK-1 phosphorylation (Figure 4)
,
consistent with JNK activation via Ras.53
These findings
demonstrate the activation of a second signaling pathway dependent on
FGF-2 after endothelial injury.
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Denudation of an endothelial monolayer leads to outgrowth from the
wound edge and recoverage of the denuded zone. To determine whether
Egr-1 plays a critical role in endothelial repair, monolayers were
incubated with an antisense oligonucleotide targeting a specific
15-base sequence unique to Egr-1 mRNA before injury. This oligomer,
E11, was synthesized with phosphorothioate rather than phosphodiester
linkages to increase resistance to possible exonucleolytic
cleavage54,55
and does not bear the guanine quartet motif
that can reportedly interfere with biological processes by nonspecific
means.56-59
E11 inhibited the induction of Egr-1 by serum
at a concentration of 1 µmol/L (Figure 5A)
. Moreover, E11 blocked outgrowth from
the wound edge 72 hours after injury (Figure 5B)
. In contrast, an
identical amount of E11C, a size-matched, base-scrambled counterpart of
E11, had no effect either on the induction of Egr-1 (Figure 5A)
or
regrowth after injury (Figure 5B)
. Trypan blue exclusion experiments
revealed that E11 inhibition was not the result of toxicity (data not
shown). E11 inhibition was reversible, as withdrawal of E11 from the
medium 72 hours after injury resulted in resumption of repair and
recoverage of the denuded zone (E11F, Figure 5B
).
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S transition.60
Additionally, antisense
oligonucleotides directed at Egr-1 have been found to attenuate rat
glomerular mesangial cell proliferation61
and inhibit
excitatory synaptic transmission in rat hippocampi.62
The
present findings extend these observations by providing the first
direct link between Egr-1 and wound healing. Parallels with Vascular Injury in Vivo
Although mechanical injury of arterial endothelium can lead to local neointima formation in animal models,7,8,63 this process is less well characterized in humans. The frequency of catheter-induced lesions during coronary angiography or balloon angioplasty is rare,5,6,64 but its very occurrence suggests an important role for endothelium in the response to injury. Catheterization can denude endothelial cells from the blood vessel wall without disrupting the internal elastic lamina or underlying media65 and produce fibrocellular lesions.5 Lesion formation at sites of endothelial denudation have been described in dogs undergoing routine catheterization of the LMCA.66 As endothelial injury can trigger paracrine signaling and transcriptional activation by FGF-2 (present study), catheter-induced lesions at sites independent of pre-existing lesions may be a consequence of growth factor release from the vessel wall.
Studies using the rat carotid model implicate an early role for
endogenous FGF-2. For example, proliferation is blocked if FGF-2
antibodies are administered at the time of injury67
but not
after 4 days.68
Whether neointima formation in response to
mechanical injury is compromised in egr-169
or
FGF-270
knockout mice is presently not known. An
attenuated response to injury in these mice would complement the
present findings using oligonucleotide inhibitors of Egr-1 (Figure 5B)
.
That inducible ERK-1/2 activity after balloon injury to the rat carotid
is FGF-2 dependent and is blocked by the presence of
PD9805971
adds weight to this possibility. Our inability to
completely inhibit Egr-1 activation by targeting FGF-2 (Figure 1, C and D)
nonetheless suggests that factors other than FGF-2 per se
are involved. Elucidation of specific cell surface events that trigger
the induction of these regulatory factors after injury will provide
additional insights on the earliest molecular events underlying
cellular changes to the vessel wall.
| Acknowledgements |
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| Footnotes |
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Supported by grants from the National Health and Medical Research Council of Australia (NHMRC) and National Heart Foundation of Australia. H.C. Lowe is a recipient of a Medical Postgraduate Research Scholarship from the NHMRC. L.M. Khachigian is holder of an R. Douglas Wright Fellowship from the NHMRC.
Accepted for publication November 18, 1998.
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F. L. Day, L. A. Rafty, C. N. Chesterman, and L. M. Khachigian Angiotensin II (ATII)-inducible Platelet-derived Growth Factor A-chain Gene Expression Is p42/44 Extracellular Signal-regulated Kinase-1/2 and Egr-1-dependent and Mediated via the ATII Type 1 but Not Type 2 Receptor. INDUCTION BY ATII ANTAGONIZED BY NITRIC OXIDE J. Biol. Chem., August 20, 1999; 274(34): 23726 - 23733. [Abstract] [Full Text] [PDF] |
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E. S. Silverman and T. Collins Pathways of Egr-1-Mediated Gene Transcription in Vascular Biology Am. J. Pathol., March 1, 1999; 154(3): 665 - 670. [Full Text] [PDF] |
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