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INTRODUCTION |
Vascular endothelial growth factor
(VEGF)1 is an important
regulator of new blood vessel formation in both health and disease states. The effects of VEGF are mediated by two high affinity transmembrane receptors, FLK-1/KDR and FLT-1 (1). Of these two
receptors, FLK-1/KDR appears to play a more prominent role in
VEGF-mediated signaling. FLK-1/KDR is a membrane-bound receptor of the
tyrosine kinase family that is expressed exclusively in endothelial
cells. The FLK-1/KDR receptor is expressed early in the mouse embryo,
where it is believed to play an important role in endothelial cell
differentiation and vasculogenesis (2). In the adult, FLK-1/KDR
expression is down-regulated (3). However, the gene may be
reactivated and/or up-regulated in tumor vascular beds or in the
setting of cardiac angiogenesis (4) and proliferative retinopathies (5,
6). In addition, FLK-1/KDR may serve an important role in wound healing
and bone remodeling (7).
An understanding of the mechanisms that underlie the transcriptional
regulation of the flk-1/KDR gene might provide important information about the molecular basis of endothelial cell
differentiation and angiogenesis. The human and mouse
flk-1/KDR promoters have been sequenced and characterized
(8-12). Under in vitro conditions, the 5'-flanking region
and first exon have been shown to contain information for endothelial
cell-specific expression, whereas in transgenic mouse assays, intronic
enhancer sequences play a critical role in mediating expression within
the vasculature (13).
Although the above studies provide insight into the mechanisms of
endothelial cell-specific gene regulation, they do not address the
question of how the flk-1/KDR promoter is temporally
controlled by positive and negative regulators of angiogenesis.
Transforming growth factor-
1 (TGF-
1) has
a biphasic effect on basic fibroblast growth factor- and
VEGF-induced angiogenesis in vitro (14). At low
concentrations TGF-
1 enhances endothelial cell response, whereas at high concentrations, TGF-
1 inhibits the
effects of angiogenic factors such as VEGF or basic fibroblast growth
factor on endothelial cells (14, 15). In a previous study,
TGF-
1 was shown to down-regulate expression of
flk-1/KDR at the level of mRNA, total protein, and
125I-VEGF binding capacity (16). These findings raised the
interesting possibility that TGF-
1 exerts its
anti-angiogenic effect, at least in part, through the inhibition of the
VEGF signaling pathway. In this study, we extend these observations by
showing that TGF-
1 suppresses flk-1/KDR
expression through a palindromic GATA site in the 5'-untranslated
region (5'-UTR).
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Bovine aortic endothelial cells (BAEC)
(Clonetics Corp.) and human embryonic kidney (HEK) 293 cells (American
Type Culture Collection CRL-1573) were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS). BAEC were used within the first 10 passages.
Plasmids--
For construction of the 2.2-kb
flk-1-luc plasmid, a region spanning
1940 to +296 bp of
the human promoter was generated by exonuclease III digestion of
the full-length flk-1/KDR promoter (generously provided by
Cam Patterson, University of Texas Medical Branch, Galveston, TX) and
cloned into the pGL2-basic vector (Promega). To generate the
KpnI-luc construct, the 2.2-kb flk-1-luc plasmid was digested with KpnI, releasing a fragment spanning region
1940 to
115. The remaining vector was religated, resulting in a
plasmid that contained
115 to +296 of the flk-1/KDR
promoter coupled to luciferase. A series of internal deletions and
point mutations were introduced into the flk-1/KDR promoter
by polymerase chain reaction methodology. To generate
KpnI(
GATA)-luc, two polymerase chain reaction fragments
(A and B) were amplified from 2.2-kb flk-1-luc. Fragment A
spanned a region between the KpnI site at
115 and +102,
whereas fragment B spanned a region between +187 and the
XhoI site of pGL2-basic. The primers were designed in such a
way as to introduce an NdeI site in the deleted region. Fragment A was digested with KpnI and NdeI,
whereas fragment B was digested with XhoI and
NdeI. Fragments A and B were then inserted into
KpnI/XhoI-digested 2.2-kb flk-1-luc in
a three-way ligation. The resulting plasmid contained a deletion of
flk-1/KDR sequences between +103 and +187. A similar
strategy was used to generate deletions of the Sp1 and NF-
B sites
(KpnI(
Sp1/NF-
B)-luc), resulting in a plasmid that
contained a deletion of flk-1/KDR sequences between
99 and
60. The same polymerase chain reaction strategy was employed to
introduce the following mutations into the GATA (KpnI(GATA
mut)-luc), Sp1 (KpnI(Sp1 mut)-luc), and NF-
B
(KpnI(NF-
B mut)-luc) motifs: Sp1 mut, GGGCGG
GTTCGG, CCGCCC
CCGTTC; NF-
B mut,
GGAGAGCCCC
AAAGAGCCTT; and GATA mut,
GGATATCC
GTTTAAGC. All deletions and
mutations were confirmed by automated DNA sequencing. To generate
pGEM-bflk, a 266-bp bovine flk-1/KDR cDNA fragment was
polymerase chain reaction-amplified from reverse-transcribed BAEC total
RNA and subcloned into the pGEM-T-easy vector (Promega). Similarly,
pGEM-bGAPDH was derived by ligating a 371-bp bovine GAPDH cDNA
fragment into pGEM-T-easy.
RNA Isolation and RNase Protection Assays--
BAEC were grown
to confluence in 100-mm culture dishes, at which point the culture
medium was replaced with serum-starved medium (Dulbecco's modified
Eagle's medium plus 0.5% FBS). 24 h later, BAEC were incubated
with 10 ng/ml TGF-
1 (Peprotec) or with serum-starved
medium alone. 24 h following TGF-
1 treatment, total
RNA was purified using the Trizol reagent (Life Technologies, Inc.).
For in vitro transcription, flk-1/KDR- and
GAPDH-specific 32P-labeled riboprobes were synthesized from
pGEM-bflk and pGEM-bGAPDH, respectively. pGEM-bflk contains a 266-bp
fragment of bovine flk-1, whereas pGEM-bGAPDH contains a
371-bp fragment of bovine GAPDH from +1 to +371 (see below). Both
riboprobes were synthesized using T7 RNA polymerase (Ambion Inc.) and
purified with a Sephadex G-50 spun column (Amersham Pharmacia
Biotech). RNase protection assays were performed with an RPA III kit
(Ambion Inc.) according to the manufacturer's instructions.
Transfections and Analysis of Luciferase Activity--
BAEC and
HEK-293 cells were transfected using FuGENE 6 reagent (Roche Molecular
Biochemicals) as instructed by the manufacturer. BAEC (1 × 105 cells/well) or HEK-293 cells (2 × 105
cells/well) were seeded in 12-well plates 18-24 h before transfection. For BAEC transfections, 0.12 pmol of the reporter gene construct and 50 ng of a control plasmid containing the Renilla luciferase reporter gene under the control of a cytomegalovirus enhancer/promoter (pRL-CMV) (Promega) were incubated with 2 µl of FuGENE 6. For HEK-293
cell transfections, 0.05 pmol of the test construct, 50 ng of the
control plasmid, and 0.075 pmol of the GATA expression vector were
incubated with 2 µl of FuGENE 6. 24 h later, the cells were
washed with phosphate-buffered saline and cultured for 12 h in
Dulbecco's modified Eagle's medium plus 0.5% FBS. The cells were
then incubated in the presence or absence of TGF-
1 for
24 h, at which time they were lysed and assayed for luciferase
activity using the dual-luciferase reporter assay system (Promega) and a Lumat LB 9507 luminometer (Berthold).
To study the effect of cell proliferation on flk-1/KDR
promoter activity, BAEC (1 × 105 or 3 × 104 cells/well) were seeded in 12-well plates 18 h
before transfection and assayed for luciferase activity 48 h later
at a time when the cells were either pre-confluent or post-confluent,
respectively. To determine the effect of serum starvation on promoter
activity, transfected BAEC were washed with phosphate-buffered saline
24 h following transfection, incubated in Dulbecco's modified
Eagle's medium containing 0.5% FBS, and assayed for luciferase
activity 18 h later.
Nuclear Extracts and Electrophoretic Mobility Shift
Assays--
Nuclear extracts were prepared as previously described
(17). Double-stranded oligonucleotides were labeled with
[
-32P]dCTP and Klenow fragment and purified on the
spun column. 15 µg of nuclear extract was incubated with 10 fmol of
32P-labeled probe, 2 µg of poly(dI-dC), 2.5 fmol of
ZnSO4, and 3 µl of 10× binding buffer (100 mM Tris HCl (pH 7.5), 50% glycerol, 10 mM
dithiothreitol, and 10 mM EDTA) for 30 min at room
temperature and then at 4 °C for a further 30 min. To test the
effect of antibodies on DNA-protein binding, nuclear extracts were
preincubated with anti-GATA-2 antibody (Santa Cruz Biotechnology and a
generous gift from Dr. Stuart Orkin) or anti-Sp1 antibody (Santa Cruz
Biotechnology) for 1 h at 4 °C. In competition studies, a 10-, 50-, or 200-fold molar excess of unlabeled wild-type or mutant
oligonucleotide was added to the reaction mixture. DNA-protein
complexes were resolved on a 5% nondenaturing polyacrylamide gel
containing 5% glycerol in 0.5× buffer containing 50 mM
Tris, 50 mM boric acid, and 1 mM EDTA. The
loaded gel was fixed with 10% methanol and 10% acetic acid and then autoradiographed.
DNase I Footprint Analysis--
DNase I footprint assays were
carried out as previously described (18). A DNA fragment spanning
region
111 to +296 of the human flk-1/KDR gene was
isolated by digesting the 2.2-kb flk-1-luc plasmid with
KpnI and XhoI. The 3'-end of the coding strand
was filled in with [
-32P]dCTP and Klenow enzyme. 8 fmol of the labeled probe was mixed with 60 µg of nuclear extracts
and digested with DNase I (Takara Biochemicals, Inc.) at room
temperature for 2 min. The samples were loaded on 6% denaturing
polyacrylamide gels. The loaded gel was fixed with 10% methanol and
10% acetic acid and then autoradiographed.
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RESULTS |
TGF-
1-mediated Down-regulation of flk-1/KDR
mRNA--
Our first goal was to confirm whether
TGF-
1 down-regulates expression of flk-1/KDR
in bovine aortic endothelial cells. To this end, we employed RNase
protection assays with a probe that is specific for the bovine
flk-1/KDR gene and total RNA derived from control and
TGF-
1-treated cells. As shown in Fig.
1 (A and B), the
incubation of BAEC in the presence of 10 ng/ml TGF-
1 for
24 h resulted in a 60% reduction in flk-1/KDR
mRNA.

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Fig. 1.
Effects of
TGF- 1 on flk-1/KDR
mRNA levels. A, confluent BAEC were
serum-starved and then incubated in the absence or presence of 10 ng/ml
TGF- 1 for 24 h, at which time total RNA was
isolated. In RNase protection assays, an
[ -32P]UTP-labeled 384-bp bovine flk-1/KDR
riboprobe was incubated with no RNA (lane 2;
probe), yeast tRNA (lane 3), or 10 µg of total
RNA from control BAEC (lane 4; bAEC) or
TGF- 1-treated BAEC (lane 5; bAEC + TGF- ). The protected fragment (266 bp) represents the bovine flk-1/KDR
transcript (bflk-1). An [ -32P]UTP-labeled
bovine GAPDH riboprobe (bGAPDH) was hybridized with total
RNA as an internal control. B, shown is the quantitation of
RNase protection assays. Densitometry was used to calculate the ratio
of flk-1/KDR and GAPDH signals (Arbitrary expression
level). S.D. values were derived from three independent
experiments. b, bases.
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TGF-
1-mediated Down-regulation of the flk-1/KDR
Promoter--
We next wished to determine whether the effect of
TGF-
1 on flk-1/KDR mRNA expression was
mediated by the flk-1/KDR promoter. BAEC were transiently
transfected with the 2.2-kb flk-1-luc plasmid, which
contains a 2.2-kb region of the human flk-1/KDR promoter (between
1960 and +296) coupled to the luciferase reporter gene. Transfected BAEC were grown in the absence or presence of
TGF-
1 and assayed for luciferase activity 24 h
later. As shown in Fig. 2A,
TGF-
1 resulted in a dose-dependent reduction
in reporter gene activity, with maximal suppression occurring at a
concentration of 10 ng/ml. In subsequent studies, we demonstrated that
a promoter fragment spanning region
115 to +296 (KpnI-luc)
contained the information for TGF-
1-mediated
down-regulation (data not shown). Finally, to determine whether the
effect of TGF-
1 on flk-1/KDR promoter
activity was mediated by changes in the cell cycle, we compared
reporter gene activity in pre-confluent and post-confluent BAEC as well
as in serum-replete and serum-starved BAEC. As shown in Fig. 2
(B and C), luciferase activity did not vary
between these conditions, arguing against a direct effect of cell
proliferation on flk-1/KDR promoter activity.

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Fig. 2.
Effects of
TGF- 1 on the flk-1/KDR
promoter. A, BAEC were transiently transfected
with 0.12 pmol of 2.2-kb flk-1-luc and exposed to increasing
concentrations of TGF- 1 for 24 h. Similar
results were obtained with the KpnI-luc plasmid (data not
shown). The results show the means ± S.D. of luciferase light
units (relative to untreated cells) obtained in duplicate from three
independent experiments. Luciferase light units are corrected for
transfection efficiency as described under "Experimental
Procedures." B, BAEC were transiently transfected with the
KpnI-luc plasmid and assayed for luciferase activity at
pre-confluence or post-confluence as described under "Experimental
Procedures." The results show the means ± S.D. of luciferase
light units (relative to pre-confluent cells) obtained in triplicate
from three independent experiments. C, BAEC were transiently
transfected with KpnI-luc and incubated in 10 or 0.5% FBS
as described under "Experimental Procedures." The results show the
means ± S.D. of luciferase light units (relative to 10%
FBS-treated cells) obtained in triplicate from three independent
experiments.
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TGF-
1-mediated Down-regulation of the flk-1/KDR
Promoter Is Mediated by the GATA Motif--
The human
flk-1/KDR promoter contains a number of consensus binding
sites, including Sp1, NF-
B, and GATA (8). Previous studies have
implicated a role for the Sp1- and NF-
B-binding sites, but not the
5'-UTR GATA motif, in mediating constitutive expression of the gene (8,
12). To test whether one or more of these elements were involved in
transducing the TGF-
1 signal, the Sp1, NF-
B, and GATA
elements were deleted and/or mutated, and the resulting plasmids were
transfected into BAEC. A combined deletion of the NF-
B site and two
upstream Sp1 sites (KpnI(
Sp1/NF-
B)-luc) resulted in
significant reduction (3.8-fold) in basal promoter activity (Fig.
3). A 4-bp mutation of the NF-
B site
(KpnI(NF-
B mut)-luc) also resulted in a 3.7-fold
reduction in promoter activity, whereas a 2-bp mutation of the two
upstream Sp1 sites (KpnI(Sp1 mut)-luc) resulted in only a
10% reduction in promoter activity. These latter findings are
consistent with previously published studies (12). However, to our
surprise, a 4-bp mutation of the GATA site in the 5'-UTR
(Kpn I (GATA mut)-luc) resulted in significant reduction
(4.4-fold) in reporter gene activity, suggesting that the GATA element
is in fact important for basal expression (Fig. 3).

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Fig. 3.
Role of the 5'-UTR palindromic GATA site in
mediating TGF- 1 response.
BAEC were transfected with 0.12 pmol of each plasmid and exposed to 10 ng/ml TGF- 1 for 24 h. Every construct was
cotransfected with 50 ng of pRL-CMV to normalize the transfection
efficiency. Promoter activity units were determined as follows:
(firefly luciferase activity/Renilla luciferase) × 1000. Data are the mean of three experiments in triplicate.
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TGF-
1-mediated down-regulation of 2.2-kb
flk-1-luc and KpnI-luc was preserved in
constructs that contained a deletion or mutation of the Sp1 and/or
NF-
B sites (Fig. 3, compare
TGF-
with
+TGF-
). In contrast, a deletion or mutation of the 5'-UTR GATA palindrome completely abrogated the inhibitory response (Fig. 3).
Taken together, these results suggest that the palindromic GATA site in
the 5'-UTR of the flk-1/KDR gene is necessary for basal
expression and TGF-
1-mediated inhibition of
flk-1/KDR promoter activity.
TGF-
1-mediated Down-regulation of the flk-1/KDR
Promoter Is Associated with an Inhibition of GATA Binding--
The
above results raised the possibility that TGF-
1 inhibits
flk-1/KDR expression by interfering with GATA binding to the 5'-UTR. To test this hypothesis, we performed electrophoretic mobility
shift assays in which nuclear extracts derived from untreated and
TGF-
1-treated BAEC were incubated with a 5'-UTR GATA
probe encompassing the putative palindromic GATA site (+98 to +122) (Fig. 4A). As shown in Fig. 4
(B-D), incubation of nuclear extract from untreated BAEC
with the 32P-labeled probe resulted in the appearance of
two specific DNA-protein complexes (open and closed
arrows). The DNA-protein complexes were inhibited by the addition
of a 10-200-fold molar excess of unlabeled self-competitor (Fig. 4,
B, lane 3; and C, lanes
3-5). Moreover, the complexes were significantly inhibited by the
addition of an oligonucleotide probe competitor containing a GATA motif from the human endothelin-1 (hET-1) promoter (Fig. 4C,
lanes 6-8). In contrast, DNA-protein binding was unaltered
in the presence of a 200-fold molar excess of unlabeled probe
containing a 4-bp mutation of the flk-1/KDR GATA sites (Fig.
4B, lane 4). Of the various members of the GATA
transcription factor family, GATA-2 is believed to play the most
prominent role in endothelial cells. To determine whether the DNA
complex contained GATA-2 protein, the binding reactions were incubated
with anti-GATA-2 antibodies. The addition of an anti-GATA-2
antibody from Dr. Stuart Orkin resulted in a supershift of the upper
DNA-protein complex (Fig. 4B, lane 5, asterisk),
whereas the addition of an anti-GATA-2 antibody from Santa Cruz
Biotechnology inhibited formation of both DNA-protein complexes
(lane 6). In contrast, incubation with an antibody against
Sp1 had no effect (Fig. 4B, lane 7). Taken together, these data suggest that the 5'-UTR palindromic GATA site
binds to GATA-2.

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Fig. 4.
Interaction between the 5'-UTR palindromic
GATA site and GATA-binding protein. A, shown is
a schematic representation of the probe sequences used in
electrophoretic mobility shift assays. The palindromic GATA site and
the mutated bases are represented by lines and
asterisks, respectively. B, electrophoretic
mobility shift assays were performed with 15 µg of nuclear extracts
from BAEC incubated in the presence (+TGF- ) or absence
( TGF- ) of TGF- 1. The
open and closed arrows indicate specific
DNA-protein complexes. In competition assays, a 200-fold molar excess
of unlabeled 5'-UTR wild-type (lanes 3 and 9) or
mutant (lanes 4 and 10) GATA probe was added to
the reaction mixture. To test the effect of antibodies on the
DNA-protein complexes, antibody against GATA-2 obtained from Dr. Stuart
Orkin (lanes 5 and 11) or from Santa Cruz
Biotechnology (lanes 6 and 12) or antibody
against Sp1 (lane 7 and 13) was added to the
reaction mixture. C, electrophoretic mobility shift assays
were performed as described for B. In competition assays, a
10-, 50-, or 200-fold molar excess of unlabeled 5'-UTR GATA probe
(lanes 3 and 9, lanes 4 and
11, and lanes 5 and 12, respectively)
or hET-1 probe (lanes 6-8, respectively) was added to the
reaction mixture. D, electrophoretic mobility shift assays
were performed with 15 µg of nuclear extract from untreated BAEC and
a 32P-labeled 5'-UTR GATA probe (lanes 1 and
2), a GATA mutant (mut) probe (lanes 3 and 4), or a hET-1 probe (lanes 6 and
7). The arrows indicate specific DNA-protein
complexes. In competition assays, a 200-fold molar excess of unlabeled
5'-UTR GATA (lane 2), GATA mutant (lane 4), or
hET-1 (lane 7) probe was added to the reaction
mixture.
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Interestingly, the 5'-UTR GATA site consists of two overlapping motifs
on opposite DNA strands. To determine whether this palindromic sequence
binds to one or two molecules of GATA-2, electrophoretic mobility shift
assays were carried out with a probe containing a mutation of a single
GATA site. The incubation of this mutant probe with nuclear extract
resulted in a single DNA-protein complex that corresponded to the
faster migrating complex of the wild-type probe and to the specific
DNA-protein complex of the hET-1 probe (Fig. 4D). These
results suggest that the more slowly migrating complex seen only with
the wild-type 5'-UTR probe represents a complex between the palindromic
GATA site and two molecules of GATA protein.
Finally, to test the effect of TGF-
1 on GATA binding,
mobility shift assays were carried out with nuclear extracts derived from TGF-
1-treated BAEC. TGF-
1 treatment
did not result in a change in mobility pattern, but rather in a
significant (3-fold by densitometry) reduction in the intensity of the
GATA-binding complexes (Fig. 4, B, lanes 8-13;
and C, lanes 9-12). To confirm the inhibitory
effect of TGF-
1 on GATA binding, we carried out DNase I
footprint analyses with a labeled promoter fragment containing region
115 to +296 of the flk-1/KDR gene. As shown in Fig.
5, the region spanning the 5'-UTR GATA
elements was protected by nuclear protein derived from untreated BAEC,
but not from TGF-
1-treated BAEC. These results are
consistent with those of the mobility shift assays and strongly support
the conclusion that TGF-
1 inhibits binding of GATA
protein to the 5'-UTR.

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Fig. 5.
DNase I footprint analysis of the palindromic
GATA site. A fragment of the flk-1/KDR promoter
spanning region 115 to +296 was labeled at the 3'-end of the coding
strand and incubated with bovine serum albumin (BSA) or 60 µg of nuclear extract from control BAEC (bAEC) or
TGF- 1-treated BAEC (bAEC + TGF- ), and the
resulting mixture was digested with DNase I as described under
"Experimental Procedures." The GATA motif region protected from
DNase I is indicated by the rectangle. DNA size markers are
shown in the first lane.
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Transactivation of the flk-1/KDR Promoter by GATA Is Inhibited by
TGF-
1--
Having established the inhibitory effect of
TGF-
1 on GATA binding in vitro, we wished to
study the functional relevance of this interaction in vivo.
To this end, we carried out transactivation assays in which the 2.2-kb
flk-1-luc or KpnI-luc constructs were cotransfected with expression plasmids for either mouse GATA-1 (pXM-mGATA1) or human GATA-2 (pMT2-hGATA2) in HEK-293
cells. As shown in Fig. 6 (A
and B), cotransfection of GATA-1 or GATA-2 induced the
full-length flk-1/KDR promoter activity by 9.0- or 11.8-fold, respectively. Similar results were obtained with the shorter
KpnI-luc construct, suggesting that the palindromic GATA site in the 5'-UTR is sufficient for mediating this effect (Fig. 6,
A and B). Indeed, KpnI-luc constructs
containing a deletion (KpnI(
GATA)-luc) or mutation
(KpnI(GATA mut)-luc) of the 5'-UTR GATA site failed
to respond to GATA-1 or GATA-2 overexpression (Fig. 6, A and
B). Finally, when cotransfected HEK-293 cells were incubated
with TGF-
1, transactivation of the flk-1/KDR
promoter by GATA-1 and GATA-2 was attenuated by 2.3- and 4.5-fold,
respectively (Fig. 7). Together, these
findings support the conclusion that TGF-
1 suppression
of flk-1/KDR expression is mediated by the 5'-UTR
palindromic GATA sequence.

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Fig. 6.
Transactivation of the flk-1/KDR
promoter by GATA-1 and GATA-2. HEK-293 cells were
transiently transfected with 50 fmol of luciferase reporter plasmids
and 75 fmol of mouse GATA-1 expression plasmid (pXM-mGATA1;
A), human GATA-2 expression plasmid
(pMT2-hGATA2; B), or vector-alone plasmid (pXM
(A) and pMT2 (B)). The expression
levels were normalized to pRL-CMV activity and expressed as -fold
induction relative to cotransfection with vector alone. The means ± S.D. were derived from at least three separate experiments performed
in duplicate or triplicate.
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Fig. 7.
Attenuation of GATA-1- and GATA-2-mediated
transactivation of the flk-1/KDR promoter by
TGF- 1. HEK-293 cells were
transiently transfected as described in the legend to Fig. 6. Cells
were serum-starved, incubated with or without TGF- 1 for
24 h, and then assayed for luciferase activity. The expression
levels were normalized to pRL-CMV activity and expressed as -fold
induction relative to cotransfection with vector alone. The means ± S.D. were derived from at least three separate experiments performed
in duplicate or triplicate.
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 |
DISCUSSION |
VEGF-mediated signaling through the FLK-1/KDR receptor is believed
to play a critical role in angiogenesis both during development as well
as in the adult. Temporal regulation of flk-1/KDR expression within the endothelium may represent an important mechanism for modulating activity of this signaling pathway. For example, in the
embryo, flk-1/KDR is expressed in the developing
vasculature, whereas in the adult, flk-1/KDR is expressed at
sites of physiological and pathophysiological angiogenesis. An
understanding of the transcriptional control mechanisms that regulate
flk-1/KDR expression in these settings may provide important
information about the molecular control of angiogenesis.
Under in vitro conditions, the flk-1/KDR promoter
has been shown to direct endothelial cell-specific expression. Maximal
promoter activity of the human gene resides in fragment
225 to +268
(8), whereas endothelial cell-specific expression of the mouse gene is
mediated by sequences between
623 and +299 (9). There are several
conserved cis-regulatory elements in the mouse and human promoters, including Sp1-, AP-2-, NF-
B-, and GATA-binding sites (8,
9). Previous studies of the human promoter have supported an important
role for upstream AP-2, NF-
B, and Sp1 elements in mediating cell
type-specific expression in cultured endothelial cells (8, 12).
In this study, we confirmed the importance of the upstream NF-
B site
in mediating basal expression of the flk-1/KDR promoter. In
addition, we demonstrated a role for an overlapping palindromic GATA
sequence in mediating constitutive promoter activity. These results are
in sharp contrast to a previous study in which a 3-bp mutation of the
5'-UTR GATA site was shown to have no effect on expression levels (8).
The reason for this discrepancy is not clear. Both studies employed
primary cultures of bovine aortic endothelial cells. In the study by
Patterson et al. (8), the GGATATCC site was mutated to
GGTCGTCC, whereas in this study, GGATATCC was mutated to
GTTTAAGC. Despite these differences, both mutations are predicted to eliminate GATA binding to the two GATA sites. It is noteworthy that the GGTCGTCC mutation was
analyzed in a promoter that spanned
225 to +268 bp, whereas
GTTTAAGC was studied in fragment
150 to +296.
However, it is unlikely that such small differences in promoter context
would account for the disparate results. In the final analysis, our
findings strongly support a role for the 5'-UTR GATA motif in mediating
basal expression of flk-1/KDR.
Several factors have been implicated in the temporal regulation of
flk-1/KDR expression. For example, tumor necrosis factor-
has been shown to induce expression of both the endogenous
flk-1/KDR gene and the upstream promoter (19, 20), whereas
TGF-
1 inhibits flk-1/KDR mRNA levels in
endothelial cells. To gain a better understanding of the mechanisms
that control TGF-
1-mediated down-regulation of
flk-1/KDR expression, we examined the effect of
TGF-
1 on flk-1/KDR promoter activity. The
results are consistent with a model in which TGF-
1
represses flk-1/KDR expression by inhibiting the interaction
of GATA-2 with overlapping double GATA consensus sites in the
5'-UTR.
In vertebrate promoters, most GATA elements occur as single sites or as
direct repeats. Rarely do GATA sequences overlap one another on
opposite sides of the DNA (21). These palindromic motifs are unique in
that they interact with both the C- and N-terminal domains of GATA
protein, resulting in high affinity binding (21). However, to date,
palindromic GATA sequences have been shown to bind only one molecule of
GATA protein. In contrast, the results of this study suggest that the
5'-UTR GATA palindrome may complex with two molecules of GATA protein.
It is conceivable that the two molecules of GATA protein bind to the
overlapping GATA elements. Alternatively, GATA-GATA protein
interactions may result in dimerization at the GATA site (22). Finally,
we cannot exclude the possibility that a heterodimer involving the GATA
transcription factor and another protein is binding to the double GATA
motif. Regardless of the mechanism, the unique configuration and
binding properties of the 5'-UTR GATA motif may be important
determinants for both basal expression and
TGF-
1-mediated repression of the flk-1/KDR gene.
TGF-
1 may have several effects on transcriptional
pathways. TGF-
1-mediated activation of its cognate
receptor results in phosphorylation of cytoplasmic transcription
factors of the SMAD family. In addition, TGF-
1
may act indirectly through other families of transcription factors to
attenuate gene expression. In some cases, these mechanisms serve to
repress basal expression levels. For example, TGF-
1
inhibits cyclin A promoter activity by decreasing the phosphorylation
and activity of activating transcription factor-1 and cAMP-responsive
element-binding protein (23). In other cases, TGF-
1
signaling results in an inhibition of inducible gene expression. For
example, TGF-
1 antagonizes phorbol ester-mediated
transcriptional induction of matrix metalloproteinase-1 through
a TGF-
inhibitory element (24). In intestinal epithelial cells,
TGF-
1 attenuates glucocorticoid-mediated induction of
haptoglobin mRNA by inhibiting CAAT/enhancer-binding protein
binding to the proximal promoter (25).
Our report is the first to implicate the GATA family of DNA-binding
proteins in mediating the inhibitory effects of TGF-
1. The transfection and DNA-protein binding assays suggest that
TGF-
1 represses flk-1/KDR expression by
interfering with the binding of GATA protein to its cognate binding
sites in the first exon. There are several possible explanations for
this effect. First, TGF-
1 may inhibit GATA expression at
a transcriptional level. It is interesting to note that in
hematopoietic cells, neutralization of TGF-
1 results in
increased GATA mRNA (26). However, our observation that
GATA-mediated activation of the flk-1/KDR promoter is
inhibited in cotransfection assays argues against this mechanism. A
second possibility is that TGF-
1 induces the binding of
other transcription factor(s) to sequences within the vicinity of the GATA site, resulting in competitive inhibition of GATA binding. This
seems unlikely since TGF-
1 does not result in a change
in the pattern of mobility of the DNA-protein complexes. A final consideration is that TGF-
1 alters GATA activity at a
post-transcriptional level. Previous studies have shown that GATA
binding is influenced both by its phosphorylation state (27-29) and by
acetylation (30). Whether or not TGF-
1 signaling
interferes with one or more of these modifications in endothelial cells
remains to be established.