Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215
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ABSTRACT |
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The early growth response (Egr)-1 transcription factor serves to couple changes in the extracellular environment to alterations in gene expression. An understanding of the mechanisms that underlie Egr-1 gene regulation should provide important insights into how environmental signals are transduced by endothelial cells. The aim of the present study was to determine whether epidermal growth factor (EGF) induces Egr-1 expression in endothelial cells. In ECV304 cells, Egr-1 mRNA and protein levels were increased in response to EGF. In stable transfection assays, the 1,200-bp promoter of the mouse Egr-1 gene contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent pathway. The endogenous Egr-1 gene was similarly responsive to EGF in primary human umbilical vein endothelial cells, human coronary artery endothelial cells, and rat fat pad endothelial cells, but not in bovine aortic endothelial cells, calf pulmonary artery endothelial cells, or PY-4-1 endothelial cells. Together, these results suggest that the Egr-1 gene is responsive to EGF in a subset of endothelial cells.
gene regulation; growth factors; endothelium
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INTRODUCTION |
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THE ENDOTHELIAL LINING OF the vascular wall participates in a wide variety of homeostatic processes, including vasomotor control, hemostasis, and the transfer of cells and nutrients between blood and underlying tissue (7). Endothelial cell morphology and function vary among different segments of the vascular tree. Phenotypic heterogeneity is largely programmed by signals residing in the microenvironment (1, 7, 29). Indeed, endothelial cells have been shown to respond to a wide array of growth factors, cytokines, and hemodynamic forces. At the present time, the mechanisms by which extracellular signals are transduced by the endothelium are poorly defined. An understanding of the pathways that mediate phenotypic expression in endothelial cells would provide insight into the molecular basis of vascular diversity in both health and disease states.
One approach to this problem is to study the mechanisms of endothelial cell-specific gene expression. The immediate-early genes include members of the Fos, Jun, and early growth response (Egr) families. Egr-1 (also designated zif268, TIS 8, NFGI-A, Krox 24) encodes a zinc finger-containing DNA-binding protein in many cell types (5, 12, 21, 22, 27, 38, 42). Egr-1 mRNA and protein levels are upregulated in response to a wide variety of mitogenic and nonmitogenic stimuli, including peptide growth factors, shear stress, urea, and hypotonicity (2, 4, 6, 8, 15, 28, 30, 41). Egr-1 binds to 5'-GCG(G/T)GGGCG-3' consensus sequences within the promoter region of target genes, resulting in transcriptional activation or repression of the gene. Therefore, Egr-1 functions as an intermediary in signal transduction, coupling short-term changes in the environment to long-term changes in gene expression.
Egr-1 has been implicated in the transcriptional regulation of multiple
genes within the endothelium, including platelet-derived growth factor
(PDGF)-A, PDGF-B, tissue factor, transforming growth factor-
(TGF-
), and urokinase-type plasminogen activator (18, 36). However, the mechanisms by which the Egr-1 gene is itself regulated in endothelial cells remain poorly understood. The Egr-1 gene
has been shown to be induced in human umbilical arterial endothelial
cells by basic fibroblast growth factor (bFGF) and in bovine aortic
endothelial cells (BAECs) by acidic FGF (aFGF) (9, 20). In
BAECs, the response of Egr-1 to injury has been shown to be mediated by
a bFGF-dependent signaling pathway (32). Egr-1 expression
in human aortic endothelial cells is increased in response to fluid
shear stress (34). Finally, under in vivo conditions,
levels of Egr-1 protein and mRNA rapidly increase at the wound edges of
denuded rat aortic endothelium (19). Together, these
findings suggest that the Egr-1 transcription factor may be involved in
coordinating expression of environmentally dependent gene programs.
In the present study, we show that the Egr-1 gene is induced by epidermal growth factor (EGF) in both the ECV304 cell line and primary endothelial cells. Induction of Egr-1 by EGF is mediated by a protein kinase C (PKC)-independent, mitogen-activated protein kinase (MAPK)-dependent pathway that converges on the serum response elements (SREs) within the 5' flanking region of the Egr-1 promoter. Importantly, we demonstrate that the response of the Egr-1 gene to EGF and other growth factors varies among different types of cultured endothelial cells. These results suggest that the Egr-1 gene is regulated by cell type-specific signal transduction pathways.
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MATERIALS AND METHODS |
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Plasmid construction. A Sal I DNA fragment containing the 1,200-bp murine Egr-1 promoter was isolated from pEgr-1p1.2 [a generous gift from Vikas Sukhatme, Beth Israel Deaconess Medical Center (BIDMC), Boston, MA] and ligated into the Xho I site of the pGL2 basic vector (Promega, Madison, WI) to create Egr-1-Luc. The Egr-1 promoter constructs A-G and V were kindly provided by David Cohen (Oregon Health Sciences University). These latter constructs contain variable lengths of the Egr-1 promoter coupled to the luciferase reporter gene in the promoterless vector pXP2 (8). The full-length promoter construct (A) and Egr-1-Luc contain the same 1,200-bp region of the Egr-1 promoter. For stable transfection experiments, Egr-1-Luc was linearized with Sal I, whereas the Egr-1 promoter constructs A-G and V were linearized with Hind III.
The SRE heterologous promoter constructs were generated by PCR. The Egr-1-Luc plasmid served as a template, and the sense and antisense primers contained internal Kpn I and Hind III sites, respectively. The SRE-A fragment, corresponding to sequences betweenCell culture.
ECV304 cells [American Type Culture Collection (ATCC) CRL-1998] were
maintained in M199 medium supplemented with 10% fetal bovine serum
(FBS; Life Technologies, Gaithersburg, MD) at 37°C and 5%
CO2. The cells were grown to 50-60% confluence and
then placed in serum-free M199 medium. Forty-eight hours later, the cells were overlaid with growth factor-supplemented or mock-treated serum-free M199 medium (1:10 volume), and the culture plates were gently swirled. NIH/3T3 cells (ATCC CRL-1658), BAECs (kindly provided by Robert D. Rosenberg, Massachusetts Institute of Technology, Cambridge, MA), calf pulmonary artery endothelial cells (CPAECs), Py-4-1 mouse endothelial cells (a generous gift of Victoria Bautch, University of North Carolina, Chapel Hill, NC), and rat fat pad endothelial cells (RFPECs; a generous gift of Michael Simons, BIDMC,
Boston, MA) were grown in DMEM with 10% FBS. At 80-90% confluence, the cells were serum starved in DMEM containing 0.5% FBS
for 24 h and subsequently treated with growth factor-supplemented medium for the times indicated. Human umbilical vein endothelial cells
(HUVECs) and human coronary artery endothelial cells (HCAECs; Clonetics, San Diego, CA) were grown in endothelial growth medium-2-MV (EGM-2-MV) BulletKit (Clonetics) to confluence and were serum starved
in M199 medium supplemented with 0.5% FBS overnight before various
treatments with growth factors as described above for ECV304 cells.
Human recombinant EGF, PDGF-BB, PDGF-AA, PDGF-AB, aFGF, tumor necrosis
factor- (TNF-
), and TGF-
were purchased from PeproTech (Rocky
Hill, NJ). Human recombinant bFGF and vascular endothelial growth
factor (VEGF) were generous gifts of Michael Simons (BIDMC, Boston,
MA). Phorbol 12-myristate 13-acetate (PMA) was purchased from
Calbiochem (La Jolla, CA). In inhibition studies, the serum-starved
ECV304 cells were preincubated with 100 µM PD-98059 (New England
Biolabs, Beverly, MA), a specific MAPK kinase (MEK) inhibitor, for
1 h and then incubated in the absence or presence of 10 ng/ml EGF
for another 1 h. An equal amount of DMSO was added to the control plates.
Western blot analysis of Egr-1 expression. For Western blot analyses, whole cell lysates were prepared from serum-starved cells at various time points following addition of growth factor-supplemented or mock-treated medium. Cells were washed twice with cold PBS, harvested with a cell scraper, and lysed in ice-cold buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM EDTA, 1 mM dithiothreiotol, 0.5 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Boehringer Mannheim) for 1 h. The resulting lysates were centrifuged at 10,000 g for 10 min, and the supernatants were saved as whole cell lysates. Protein quantitation was carried out with the microprotein determination system (Sigma, St. Louis, MO). Forty micrograms of the whole cell lysates were separated by 10% SDS-PAGE and were electrotransferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature. The blot was incubated with primary rabbit polyclonal anti-Egr-1 IgG (1:1,000 dilution; Santa Cruz Biotechnology, CA) overnight at 4°C, followed by secondary antibody goat anti-rabbit horseradish peroxidase conjugate (1:1,000 dilution; Pierce, Rockford, IL). The blot was washed extensively between each incubation step. Peroxidase activity was visualized with an enhanced chemiluminescence (ECL) substrate system (Amersham, Arlington Heights, IL).
RT-PCR analysis of Egr-1 expression.
Total RNA was prepared from ECV304 cells and HUVECs by guanidine
thiocyanate-phenol-chloroform extraction (Stratagene, La Jolla, CA).
Random-primed first-strand cDNA was synthesized from 2 µg of total
RNA using the SuperScript preamplification system (Life Technologies)
according to the manufacturer's instructions. One-tenth of the
first-strand cDNA was then amplified by PCR with primer sets specific
for human Egr-1 (sense, 5'-CAGCAGTCCCATTTACTCAG; antisense,
5'-GACTGGTAGCTGGTATTG) (20) and -actin (sense,
5'-GTGGGCCGCTCTAGGCACCAA; antisense, 5'-CTCTTTGATGTCACGCACGATTTC)
(Clontech, Palo Alto, CA). PCR reactions were carried out with the
following cycle conditions: 95°C for 4 min, 30 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a final
incubation at 72°C for 7 min. One-fifth of the PCR products were
electrophoresed on a 1.2% agarose gel, and the PCR products were
visualized by ethidium bromide staining. For actinomycin D experiments,
ECV304 cells were pretreated with 10 µg/ml actinomycin D (Sigma) for
30 min before addition of EGF and were harvested for total RNA 1 h later.
Stable and transient transfections and luciferase assays.
For stable transfections, 107 ECV304 or NIH/3T3 cells were
electroporated with 20 µg of linearized Egr-1 luciferase constructs and a 1:10 molar ratio of Dra I-linearized pGK-neo
(13) (Gene Pulser II; 300 V and 600 µF; Bio-Rad
Laboratories). The cells were grown for 16 days in medium containing 1 mg/ml G418 (Life Technologies). Over 200 G418-resistant clones were
trypsinized and pooled, and equal numbers of cells were seeded in
12-well tissue culture plates. The ECV304 transfectants were starved in serum-free M199 medium for 48 h, whereas the NIH/3T3 transfectants were serum starved in DMEM with 0.5% FBS for 24 h before various treatments. The cells were then incubated for 2-26 h with medium containing EGF, aFGF, bFGF, PDGF-AB, PDGF-AA, PDGF-BB, TNF-, TGF-
, or VEGF at the doses indicated. In inhibition studies, the
serum-starved cells were pretreated with the MEK inhibitor PD-98059 at
a final concentration of 50 µM or a specific PKC inhibitor bisindolylmaleimide I (BIM; Calbiochem) at a final concentration of 5 µM for 30 min before addition of growth factors. Cells were harvested
for luciferase activity according to the manufacturer's instructions
(Promega). Light activity was measured in a luminometer (Lumat LB 9507;
EG&G Berthold, Germany) and was expressed as relative light units
(RLU) ± SD. All experiments were carried out in triplicate and
repeated at least four times.
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RESULTS |
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The Egr-1 gene is induced in ECV304 cells by replacement of the
medium.
Simple replacement of serum-free medium after 48 h of serum
starvation in ECV304 cells resulted in significant increases in Egr-1
mRNA and protein, with peak levels occurring between 30 and 60 min (Fig. 1, A and
B). The Egr-1 gene was also induced by the addition of 10%
volume of serum-free medium followed by gentle mixing (data not shown).
Preincubation of serum-starved ECV304 cells with a MAPK-specific
inhibitor (PD-98059) or a PKC-specific inhibitor (BIM) abolished Egr-1
induction by replacement of medium (Fig. 1C). The
lower-than-baseline level of Egr-1 protein in the PD-98059-treated
cells suggests that constitutive expression of Egr-1 is mediated by a
MAPK signaling pathway. To establish a role of the Egr-1 promoter in
transducing this response, we stably transfected ECV304 cells with the
Egr-1-Luc construct. Over 200 G418-resistant clones were pooled and
seeded at the same density in 12-well tissue culture plates. Luciferase
activity was increased 1.8-fold by simply changing the serum-deprived
medium (Fig. 1D, vs. C). The induction was abrogated by
the addition of PD-98059 or BIM. These results suggest that the
change-of-medium effect is transduced by a PKC-dependent MAPK signal
transduction pathway operating through the 5' flanking region of the
Egr-1 promoter. The sensitivity of the Egr-1 gene to these
manipulations may be explained by transient alterations in hemodynamic
forces or perhaps by subtle changes in the microenvironment of the
freshly exchanged media. Regardless of the underlying mechanism, the
results underscore the importance of including appropriate controls in
studies of Egr-1 gene regulation.
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The Egr-1 gene is induced in ECV304 cells by EGF but not by PDGF-BB
or bFGF.
Serum-deprived ECV304 cells were gently overlaid with 10% volume of
serum-free M199 medium with or without 10 ng/ml EGF, 10 ng/ml PDGF-BB,
or 40 ng/ml bFGF. Growth factor- and mock-treated cells were incubated
for 60 or 120 min and then harvested for total RNA and whole cell
protein extracts. Egr-1 mRNA and protein levels were significantly
increased in ECV304 cells by treatment with EGF but not with PDGF-BB or
bFGF, with peak levels occurring at 60 min (Fig.
2A shows Egr-1 mRNA at 60 min;
Fig. 2B shows Egr-1 protein at 60 min and 120 min). To
determine whether EGF-mediated induction of Egr-1 was dependent on new
mRNA synthesis, ECV304 cells were pretreated with 10 µg/ml
actinomycin D for 30 min before a 60-min incubation with EGF.
Actinomycin D completely abolished EGF-mediated induction of Egr-1 mRNA
(Fig. 2C), indicating that Egr-1 induction by EGF requires
de novo mRNA synthesis. The addition of actinomycin also reduced basal
levels of Egr-1 in untreated cells, suggesting a rapid turnover of
Egr-1 mRNA.
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The 1,200-bp 5' flanking region of the Egr-1 promoter confers
responsiveness to EGF in ECV304 cells.
We next tested whether the 1,200-bp Egr-1 promoter contained
information for EGF response. ECV304 cells stably transfected with
Egr-1-Luc construct were serum deprived for 48 h, incubated in the
presence or absence of growth factor-supplemented serum-free medium for
4 h, and harvested for luciferase activity. To control for the
change-of-medium effect, the medium was replaced in both treated and
untreated cells. The Egr-1-Luc reporter gene activity was induced by an
average of 3.2-fold in the presence of 10 ng/ml EGF but was unchanged
by the addition of 40 ng/ml aFGF, 40 ng/ml bFGF, 10 ng/ml PDGF-AB, 10 ng/ml PDGF-AA, 10 ng/ml PDGF-BB, 25 ng/ml TNF-, 10 ng/ml TGF-
, or
40 ng/ml VEGF (Fig. 3A).
Incubation of transfected cells with up to 100 ng/ml of PDGF-BB or bFGF
for 2, 4, 14, and 24 h had no effect on reporter gene activity
(data not shown). As expected, the Egr-1 promoter was induced by the addition of either 10% FBS (2.6-fold) or 100 nM PMA (14-fold) (Fig.
3A shows the response to 10% FBS). The Egr-1 promoter
induction by EGF was dose and time dependent (Fig. 3, B and
C, respectively). Induction was maximal (3.2-fold) at
4 h of treatment with 10 ng/ml EGF. This effect was abolished by
50 µg/ml anti-EGF polyclonal antibody (Fig. 3B). In cells
that were not exposed to EGF, basal levels of Egr-1 promoter activity
were unaffected by the addition of anti-EGF antibody (data not shown).
In summary, the 5' flanking region of the Egr-1 gene contains
information for response to EGF, serum, and PMA but not to other growth
factors.
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Progressive deletion of SREs in the Egr-1 5' flanking sequence
diminishes response to EGF in ECV304 cells.
To delineate the promoter regions responsible for mediating Egr-1
response to EGF, a series of 5' and internal deletion mutants of the
Egr-1 promoter (Fig. 4A) were
stably transfected into ECV304 cells. A promoter construct that
contains the 1,200-bp full-length Egr-1 5' flanking sequence (Egr-1-A)
was induced 2.7-fold by EGF (Fig. 4B). Deletion of the
upstream AP-1 binding sites (Egr-1-C) did not affect the level of EGF
inducibility. On the other hand, deletion of all five SREs (Egr-1-B)
from the full-length promoter resulted in a complete loss of response
to EGF. A construct that lacked the 5' SRE cluster but contained the 3'
SRE cluster (Egr-1-E) was induced a modest 1.8-fold, whereas a
construct that contained an internal deletion of the 3' SRE cluster
(Egr-1-D) was induced 2.4-fold in the presence of EGF. A construct that
contained only the most proximal of the 3' SREs (Egr-1-F) failed to
respond to EGF (Fig. 4B). The differential response of the
SRE clusters to EGF was preserved in the context of a heterologous
promoter. When coupled to the human vWF core promoter, the 5' SRE
cluster was induced 2.2-fold, whereas the 3' SRE cluster was induced
only 1.4-fold with EGF (Fig. 5; compare
SRE-A-Luc with SRE-B-Luc). A construct that contained both the 5' and
3' clusters (SRE-C-Luc) was induced 2.5-fold by EGF (Fig. 5). The
vWF-90-Luc construct by itself did not respond to EGF or to other
growth factors in ECV304 stable transfectants (data not shown). These
results suggest that while the 3' SRE cluster in the Egr-1 promoter
contributes to EGF inducibility, the 5' SRE cluster is more efficient
in transducing the EGF signal.
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Egr-1 induction by EGF in ECV304 cells is mediated by the MAPK
pathway.
To elucidate the signaling pathways that mediate EGF inducibility of
Egr-1 in ECV304 cells, Egr-1-Luc stable transfectants were treated with
protein kinase inhibitors. ECV304 Egr-1-Luc stable transfectants were
pretreated with 50 µM PD-98059 or 5 µM BIM for 30 min and then
incubated with serum-free medium in the absence or presence of 100 nM
PMA or 10 ng/ml EGF for another 4 h. Basal reporter gene activity
was downregulated by the addition of PD-98059. These results are
consistent with those of the endogenous gene (see Figs. 1C
and 6B) and suggest that
constitutive Egr-1 expression requires MAPK-dependent signaling.
EGF-mediated induction of the Egr-1 promoter was completely abolished
by PD-98059, whereas the PKC inhibitor BIM had no effect (Fig.
6A). In contrast, PMA-mediated induction of the Egr-1
promoter was abrogated both by PD-98059 and BIM (Fig. 6A).
EGF-mediated induction of the endogenous Egr-1 gene was also abolished
by 100 µM PD-98059 in ECV304 cells (Fig. 6B). These
results suggest that Egr-1 induction by EGF in ECV304 cells is mediated
by a PKC-independent MAPK signaling pathway. To provide functional
support for these observations, an Elk-1 expression vector was
cotransfected with an Elk-1-responsive luciferase reporter gene
construct in ECV304 cells. Treatment with EGF, but not with PDGF-BB or
bFGF, resulted in increased Elk-1 transactivation activity (Fig.
6C). These results imply that EGF-mediated activation of
MAPK is coupled to an increase in Elk-1 activity.
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Differential Egr-1 gene regulation in endothelial cells.
In the next set of experiments, we determined whether EGF
responsiveness of the Egr-1 gene is preserved in other endothelial cell
types or whether the pattern of growth factor-mediated response varies
among different endothelial cell types. Serum-starved HUVECs were
growth factor or mock treated for 60 min and subjected to RT-PCR and
Western blot analyses. Egr-1 protein and mRNA levels were induced by
EGF and bFGF but not by PDGF-BB (Fig. 7,
A and B, respectively). Egr-1 protein was rapidly
induced by EGF, with peak levels occurring at 1 h and returning to
baseline after 2 h (Fig. 7C). Similarly, in HCAECs,
Egr-1 levels were upregulated by EGF and bFGF but not by PDGF-BB. In
RFPECs, Egr-1 was induced by EGF and PDGF-BB but not by bFGF. In BAECs
and Py-4-1 mouse endothelial cells, Egr-1 was induced by bFGF alone,
whereas, in CPAECs, Egr-1 was only slightly induced by PDGF-BB alone
(Fig. 7A). The response of Egr-1 to aFGF and VEGF also
varied among different endothelial cells (data not shown). Together,
these results show that the Egr-1 gene is regulated by endothelial cell subtype-specific mechanisms.
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The Egr-1 gene is induced in NIH/3T3 cells by EGF, PDGF-BB, and
bFGF.
The Egr-1 gene is expressed in a variety of nonendothelial cell types.
To compare the pattern of response in endothelial and nonendothelial
cells, NIH/3T3 cells were serum starved in 0.5% FBS medium for 24 h and treated with EGF, PDGF-BB, and bFGF as described for ECV304
cells. In Western blot analyses, Egr-1 protein levels were induced in
all treated cells, with highest levels occurring in the presence of
PDGF-BB and bFGF (Fig. 8A). To
determine whether the 1,200-bp Egr-1 promoter has the capacity to
respond to other growth factors, NIH/3T3 cells were stably transfected with the Egr-1-Luc construct. Pooled transfectants were serum starved
in 0.5% FBS for 24 h and incubated with various growth factors
for 4 h, and the luciferase activity was assayed. Under these
conditions, reporter gene activity was induced 6.6-fold by bFGF,
7.6-fold and 8.3-fold by PDGF-BB and PDGF-AB, respectively, and
3.4-fold by EGF (Fig. 8B). Together with the results of the ECV304 cells and other endothelial cells, these findings suggest that
the Egr-1 gene is regulated in a cell type-specific fashion.
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DISCUSSION |
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Far from being a passive barrier, the endothelium plays an active role in many homeostatic processes. Endothelial cells respond to signals residing in the local microenvironment and thereby adapt to the spatially and temporally diverse needs of the underlying tissue. Endothelial cell response involves the activation of intracellular signaling pathways, alterations in gene expression patterns, and changes in cellular phenotype. Because the Egr-1 gene lies at the convergence of a wide array of signals and in turn mediates the expression of multiple gene products, an understanding of its regulation may provide insight into how extracellular signals modulate gene expression and ultimately lead to changes in cell phenotype.
In the present study, the Egr-1 gene was shown to be induced by EGF in ECV304 cells and in a subset of primary endothelial cells. EGF-mediated induction of Egr-1 was conferred by DNA sequences within the 1,200-bp promoter region. The 5' flanking region of the Egr-1 gene contains five SREs that are organized into two discrete clusters, a 5' cluster that contains SREs 3, 4, and 5, and a 3' cluster that contains SREs 1 and 2. As originally defined in the context of the c-fos promoter, the SRE consensus sequence, CC(A/T)6GG, is recognized by the serum response factor (SRF), whereas neighboring Ets motifs form a complex with the ternary complex factor (TCF). In response to extracellular signals, the SRF and TCF bind to their cognate DNA elements and enhance transcription. Each of the SRE consensus sites within the Egr-1 promoter has been shown to be a functional SRE and to compete with the c-fos SRE sequences for binding by nuclear proteins (6). However, our findings suggest that EGF-mediated transactivation of the Egr-1 gene is mediated primarily by the 5' SRE cluster (SREs 3, 4, and 5). The same cluster of SREs has been shown to mediate response to shear stress and urea in HeLa and medullary collecting duct cells, respectively (8, 34). In contrast, the 3' SRE cluster has been implicated in granulocyte macrophage colony-stimulating factor-mediated induction of the Egr-1 gene (26, 31). Finally, all five SREs play a more equal role in transducing the PDGF-BB response in rat mesangial cells (30). Together, these observations suggest that the SREs in the upstream promoter region of the Egr-1 gene may cooperate in various combinations to transduce extracellular signals.
To our knowledge, the present study is the first to show that Egr-1 is responsive to EGF in endothelial cells. EGF, a 53-amino acid peptide, is a member of a large family of EGF receptor ligands that includes amphiregulin (35), heparin-binding EGF (14), and epiregulin (40). EGF upregulates the expression of genes involved in the adhesion, attachment, migration, and proliferation of many cell types. Transcriptional activation is mediated by a variety of EGF-responsive elements, including SREs (11), Sp1 (25), cAMP-response element (CRE) (24), and a 22-bp palindromic EGF response element (ERE) sequence (43). In endothelial cells, EGF signaling has been shown to play a role in angiogenesis. For example, under in vitro conditions, the proliferative and migratory responses of endothelial cells are stimulated by EGF (3, 44). These effects have been associated with increased levels of the Ets-1 transcription factor (17). In in vivo models, the EGF peptide has been shown to be integrally involved in the angiogenic process (33, 37). Together, these studies support an important role for EGF in the endothelial cell function. The results of the present investigation suggest that, in endothelial cells, the effect of EGF may be coupled to the activation of the immediate early gene Egr-1 and a downstream cascade of Egr-1 responsive genes.
ECV304 cells were reported to have originated from HUVECs by spontaneous transformation (39) and have been shown to retain many properties of endothelial cells, including the capacity to respond to VEGF (23) and to form capillary-like tubular structures in matrigel systems (16). However, in contrast to HUVECs, ECV304 cells do not express the vWF and CD31 antigens and display autonomous growth in the absence of growth factor-supplemented medium (16, 39). This latter property is presumably coupled to changes in growth factor receptor-mediated signaling pathways and may account for the loss of bFGF response of the Egr-1 gene in ECV304 cells. More recently, molecular genetic methods have uncovered evidence of cell line cross-contamination involving the ECV304 cell line (10). Until the precise nature of the ECV304 cell line is resolved, results derived from this cell type must be interpreted with caution and confirmed in primary endothelial cells.
The above limitations notwithstanding, our results clearly show that the Egr-1 gene is induced by EGF in some but not all cultured endothelial cells. Moreover, the response to PDGF-BB and bFGF also differed among endothelial cells. These differences are not fully explained by the species of origin. For example, bovine aortic and calf pulmonary artery endothelial cells displayed unique patterns of growth factor-mediated Egr-1 response. Our findings raise the question of whether the cell subtype-specific transcriptional networks reflect physiologically important differences or whether they are artifacts of the in vitro system. On the one hand, it is possible that diverse signal transduction networks within the endothelium are preserved when endothelial cells are uncoupled from their native tissue environment. On the other hand, cell type-specific differences in the mechanisms of Egr-1 gene regulation may arise from variable degrees of phenotypic drift. In other words, the expression of cell surface receptors and downstream signal transduction networks may be modified at different rates in different types of cultured endothelial cells.
Regardless of the underlying mechanism, our findings have wider implications for the interpretation of in vitro-based endothelial cell studies. In many cases, the response of an endothelial cell, whether to growth factors, to hemodynamic factors, or to hypoxia, is assumed to be a representative property of all endothelial cells. The observation that Egr-1 expression in endothelial cells from different sources responds to different growth factors is a clear example of how the results in one endothelial cell type do not necessarily apply to other cell subtypes. Data that are derived from in vitro studies of endothelial cells should be qualified according to the nature of the assays as well as the origin of the endothelial cell.
On a final note, we have recently shown that systemic administration of EGF to mice results in the upregulation of the Egr-1 gene in restricted populations of endothelial cells (23a). Together with the findings of the present investigation, these results suggest that the Egr-1 gene is indeed regulated by cell subtype-specific mechanisms in vivo. Rather than serving a common function throughout the endothelium, the Egr-1 gene may serve to regulate target gene expression according to the unique demands of the local tissue environment.
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ACKNOWLEDGEMENTS |
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We thank Vikas Sukhatme for helpful suggestions. We also thank David Cohen for providing the Egr-1 constructs.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-60585-01. J. C. Tsai is the recipient of a Judith Graham Pool Postgraduate Research Fellowship Award from the National Hemophilia Foundation. W. C. Aird is the recipient of an American Society of Hematology Scholar Award.
Address for reprint requests and other correspondence: W. C. Aird, Molecular Medicine, RW-663, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston MA 02215 (E-mail: waird{at}caregroup.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 June 1999; accepted in final form 19 May 2000.
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