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INTRODUCTION |
Endothelin-1 (ET-1)1 is
a 21-residue peptide synthesized and secreted by vascular endothelial
cells. It is a member of a family of structurally related peptide
hormones and is the most potent endogenously produced vasoconstrictor
known (for review, see Ref. 1). The enhanced secretion of ET-1 during
myocardial ischemia has been linked with enhanced contractility in the
failing heart (2) as well as with the progression of heart failure (3). The combined actions of ET-1 and the endothelial cell relaxing factor
nitric oxide may be important in regulating vascular tone and blood
pressure (1). In addition to its vasomodulating activity, ET-1 has been
shown to modulate multiple cell functions including proliferation,
proto-oncogene and protein kinase activity (1), induction of inotropy
and hypertrophy in cardiac muscle, and activation of cardiac specific
genes (4, 5). At least two cell surface receptors for the endothelins
are present on multiple cell types, and most of the actions of the
peptides are probably relayed through these receptors (for review, see
Ref. 6).
Expression of the ET-1 gene in endothelial cells is subject to complex
regulation by numerous factors, including but probably not limited to
thrombin (7), transforming growth factor-
(8), shear stress (9, 10),
tumor necrosis factor-
(11), interleukin-1 (12), insulin (13),
angiotensin II (14), nitric oxide (15), and hypoxia (15-20). Molecular
analyses of the gene have revealed cis-acting sequences that
contribute to ET-1 expression (21). The promoter region contains
activator protein-1 (AP-1), GATA-2, nuclear factor-1 (NF-1), and
acute phase regulatory element sites upstream from classical CAAT and
TATAA boxes. The AP-1 sites have been shown to mediate transcriptional
induction by Fos and Jun proteins (22) as well as the cellular response
to phorbol esters (13) and thrombin (7). The GATA-2 and AP-1 elements
are essential for high level expression of ET-1 in endothelial cells
(23). In addition to the 5'-elements, the ET-1 gene also contains two AUUUA sequences in the 3'-untranslated region which probably determine the short (15-20 min) half-life of the ET-1 message and its induction by protein synthesis inhibitors (21).
Hypoxia is one of the most potent inducers of ET-1 gene expression in
endothelial cells and may be the primary cause of the increased
production of ET-1 during myocardial ischemia (24-27). Hypoxia induces
the synthesis and secretion of ET-1 in isolated endothelial cells by a
mechanism that is antagonized by nitric oxide and carbon monoxide and
mimicked by transition metals (15-17). The ET-1 promoter contains an
inverted hypoxia-inducible factor-1 (HIF-1) binding site at position
118 base pairs upstream of the transcription start site which binds
the factors HIF-1
and ARNT (HIF-1
) and is essential for the
promoter response to hypoxia (20, 28). Here we report that this
response also requires three adjacent transcription factor binding
sites to form a functional hypoxia-responsive complex. The response is
endothelial cell-specific and is modulated but not necessarily
dependent on interactions with the activator protein
p300/CBP.
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MATERIALS AND METHODS |
Cell Culture--
Human umbilical venous endothelial cells
(HUVECs) were prepared from the umbilical cords of multiple donors as
described previously (28). Briefly, umbilical cord veins were rinsed
twice with phosphate-buffered saline and filled with 0.1%
collagenase in phosphate-buffered saline containing Ca2+
and Mg2+. After incubation at 37 °C for 15 min, gentle
flushing of the vein released a suspension of isolated cells. These
were seeded onto gelatin-coated culture dishes, and the adherent ECs
were cultured in M199 medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, L-glutamine, and 10% human
serum. In some experiments HUVECs were purchased from Clonetics (San
Diego, CA) and cultured as described previously (19). HeLa, HepG2, and human embryonic kidney (HEK)-293 cells were also cultured as described previously (29).
Hypoxia--
Our conditions for hypoxic incubations have been
described in detail previously (19, 28, 30). Briefly, cells were
incubated in a temperature- and humidity-controlled environmental
chamber in an atmosphere containing 0.5% O2, 5%
CO2, balance N2. Oxygen tension inside the
chamber was monitored continuously with an oxygen-sensitive electrode
(Kathaerobe Controls, Philadelphia). All cell manipulations including
media changes and harvesting took place under hypoxia to avoid
transitory reoxygenation.
Plasmid Constructs and Site-directed Mutagenesis--
ET-1
promoter deletions have been described previously (13, 28, 31). Our
methods for site-directed mutagenesis and mutation of the HIF-1 binding
site were also described previously (28). Mutations of the AP-1,
GATA-2, and NF-1 binding sites in the
176 truncations were introduced
by the same polymerase chain reaction-based procedures. The primers
were GATA-2: 5'-CCGACTCCGGCTGCACGTTGCCTGTTGGTGACTAATA AC-3'; AP-1:
5'-GACACCTAATAACACAATAACATTGTCTGGGGCTGG-3', with the appropriate
opposite strand primers; and NF1: 5'-AACAACATTGTCTGGGGCTGGAAT3-' and
5'-ACCTTATTAGTCACCAACAGGCAACGT-3'. These reactions replaced the
bases TTAT, GTGA, and CAAT in the consensus GATA-2, AP-1, and NF-1
sites with CCGA, ACAC, and TACC, respectively. All mutations were
confirmed by sequencing. Other vectors used in transfection and
infection analyses including E1A clones, p300,
pGL3HREEpo, pRSV-luciferase,
pEnoHRE, and adenoviral vectors expressing E1A have
been described previously (32).
Plasmid Insertions--
A 70-base pair fragment from the
enolase-1 promoter containing the enolase-1 HRE (33) was inserted into
the BamHI site of pET-1Luc-WT (both
176 and -669 promoters) by blunt end ligation. Spacer DNA sequences containing 50 base pairs were inserted in between GATA-2 and HIF-1, and HIF-1 and
AP-1, respectively, by polymerase chain reaction-based insertional
mutagenesis using the same methods described above for site directed
mutagenesis with the foreign sequence overhanging 5'- and 3'-primers of
36 and 30 base pairs each complementary to the respective site for insertion. The sequence of the inserted DNA was
5'-ATGCTAGGCGTCATGAGTACGAGGTCGGAGCTACGTACTGCCGTTGTACG-3'. All
new constructs were confirmed by sequencing.
Northern Blot Analyses--
RNA transcript levels were measured
by Northern blots as described previously (19, 29). Briefly, total RNA
was isolated by solubilizing cells on the plate in 4 M
guanidinium thiocyanate (0.25 ml/106 cells) and pelleting
through cesium chloride. Agarose gels, blotting, and hybridizations
were all as described previously (19). Complementary DNA probes
including human ET-1 (purchased from ATCC) and
-actin (29) were
labeled by random priming (Prime-It kit, Stratagene) to
108cpm/µg DNA. RNA bands on the autoradiographs were
quantitated using a UMAX Powerlook II scanner, Power Macintosh
8500/150, and Adobe software. RNA loading on gels was monitored by
ethidium staining and by probing with
-actin as the control.
Transfections--
HUVEC, HeLa, HepG2, and HEK-293 cells were
transfected by the calcium phosphate method as described previously
(19). HUVECs were transfected at 80% confluence; other cell lines were
transfected at 30-40% confluence; cells were exposed to 0.5 ml of
calcium phosphate precipitate containing 8-10 µg of plasmid DNA
including an internal control (Renilla luciferase from Promega
Biotechnology) for 8-12 h. Transfected cultures were exposed to
hypoxia after a further 24-48 h. Expression of luciferase activity,
normalized to the internal control, was determined as described
previously (19).
Nuclear Extracts and Gel Mobility Shift Assay--
Nuclear
extracts were prepared from confluent plates of HUVECs,
C2C12 myoblasts, or HeLa cells grown under a
normal aerobic environment, treated with 100 µM
CoCl2, or exposed to hypoxia as described previously (19,
28, 34). For hypoxic cell extracts, cell lysis was performed with the
cells still under hypoxia to avoid reoxygenation effects (35).
Sequences of the oligonucleotide probes used were as
follows: HIF-1Epo: 5'-AGCTTGCCCTACGTGCTGTCTCAGA-3'; ET-1HIF-1(WT): 5'-CTCCGGCTGCACGTTGCCTG-3';
ET-1HIF-1(M): 5'-CTCCGGCTTACCGTTGCCTG-3'; AP-1(WT): GCCTGTTGGTG ACTAATAAC-3'; AP-1(M):
5'-GCCTGTTGACACCTAATAAC-3'; GATA-2(WT): 5'-CCTGGCTTATCTCCGGCTGC-3';
GATA-2(M): 5'-GGCCTGGCCCGACTCCGGCT-3'; ET-1HRE(WT):
5'-GGCCTGGCTTATCTCCGGCTGCACGTTGCCTGTGGGTGACTAATCACA-3'. Site
mutations within the ET-1HRE(WT) oligonucleotide were the same as those described for the ET-1HIF-1(M)
oligonucleotide. Gel-purified double-stranded oligonucleotides were end
labeled with [32P]ATP using T4 polynucleotide kinase
(Promega) and [
-32P]ATP (NEN Life Science Products).
Equal amounts of radioactive probe (1.5-2.5 × 104
cpm) were added to binding reactions that contained 8 µg of nuclear extract protein in 20 µl of a buffer containing 4 mM Tris
(pH 7.8), 12 mM Hepes (pH 7.9), 60 mM KCl, 30 mM NaCl, 0.1 mM EDTA, 1 µg of poly(dI-dC)
(Amersham Pharmacia Biotech). Antibodies against c-Jun, GATA-2,
Sp1, c-Fos, and p300 were from Santa Cruz Biotechnology; anti-HIF-1
antibody was from Lab Vision Corp. (Freemont, CA). Reactions were
incubated for 15 min at 22 °C before separating on nondenaturing 6%
polyacrylamide gels at 4 °C (34).
In Vitro Transcription-Translation--
Full-length cDNAs
were obtained encoding c-Jun, c-Fos (both generous gifts of Tom Curran,
St. Jude Children's Research Hospital, Memphis), GATA-1 and GATA-2
(generous gifts from S. Orkin, Dana Farber Cancer Institute, Harvard
Medical School, Boston) HIF-1
(generous gift from F. Bunn, Brigham
and Women's Hospital, Harvard Medical School, Boston MA), HIF-1
(ARNT) (generous gift from Darren Richard, Institute of Signaling,
Developmental Biology and Cancer Research, CNRS, Nice Cedux 2, France).
All cDNAs were subcloned into pBlueScript SK(
) by standard
cloning procedures. The corresponding proteins were transcribed and
translated in vitro using rabbit reticulocyte lysates
(Promega).
Pull-down Assays--
Double-stranded probes biotinylated at the
5'-ends were from Life Technologies, Inc. These included the wild type
ET-1 HRE described above and individual mutations in the wild type
sequence including GATA-2(M): TTAT to CCGA; HIF-1(M): CGT to TAC; and
AP-1(M): GTGA to ACAC. Probes were also synthesized containing double
mutations of AP-1 and GATA-2, and triple mutation of all three sites.
The probes were purified by polyacrylamide gel electrophoresis, and equal amounts of complementary strands were annealed. Dynabeads M-280
Streptavidin (Dynal, Inc., Lake Success, NY) were prepared by washing
three times in phosphate-buffered saline (pH 7.4) containing 0.1%
bovine serum albumin and two times with Tris-EDTA containing 1 M NaCl. Between each wash, beads were pulled down with a
Dynal magnetic particle concentrator. Double-stranded, biotinylated oligonucleotides were added to the washed beads, and the mix was rotated for 20-30 min at 21 °C. Equal cpm of proteins translated in vitro were made to 1 × with binding buffer and
mixed with ~100 µg of Dynabeads containing 10 pmol of the
individual oligonucleotide probe in a final volume of 250 µl. The
mixture was rotated at room temperature for 20 min, and proteins bound
to the beads were separated from unbound proteins by successive washes,
three times with 0.5 × binding buffer and once with 1 × binding buffer. Higher stringency wash included two washes with 2 × binding buffer. Beads and bound proteins were pulled down with a
magnetic concentrator, suspended in 1 × sample buffer, boiled for
5 min, and resolved on SDS-polyacrylamide gels (10%) as described for
immunoprecipitation reactions.
The same procedures were used to pull down p300 from nuclear extracts.
In this case the protein eluted from the beads was electroblotted onto
nitrocellulose membranes (Bio-Rad). Blots were stained with Ponceau Red
to monitor the transfer of proteins. Membranes were blocked for 1 h at room temperature with 5% nonfat milk in TBS (25 mM
Tris, 137 mM NaCl, 2.7 mM KCl) containing
0.05% Tween 20 and incubated with anti-p300 antibody for 2-4 h in the same buffer. After washing, the blots were incubated for 1 h with a 1:7,500 dilution of horseradish peroxidase-conjugated anti-rabbit IgG
or horseradish peroxidase-conjugated donkey anti-goat IgG and
visualized using enhanced chemiluminescence (Pierce).
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RESULTS |
Functional Characterization of the ET-1 Promoter HRE--
Fig.
1A shows the sequence of the
HRE in the ET-1 promoter between base pairs
91 and
142 upstream of
the transcription start site, including the positions of transcription
factor binding sites. The base changes shown were introduced
individually and in pairs into pET1
176-Luc as described
under "Materials and Methods." The expression of the wild type
promoter and each mutation in endothelial cells cultured under aerobic
or hypoxic conditions is shown in Fig. 1A. The AP-1 and
GATA-2 mutations reduced basal expression to < 20% of the wild
type. In agreement with previous reports expression of the wild type
promoter was induced by 2.3-fold (± 0.3, n = 12;
p < 0.001) under hypoxia (28). This response was
eliminated in all constructs containing site mutations. Therefore
activation of the ET-1 promoter by hypoxia in ECs requires intact
GATA-2, HIF-1, AP-1, and NF-1 binding sites.

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Fig. 1.
Functional regulation of the ET-1 HRE.
Top panel indicates the location and sequence of the ET-1
HRE and positions of the transcription factor binding sites. Below the
diagram, base changes for each site mutation are indicated.
Bottom panels: A, relative luciferase activity of
the wild type 176 ET-1-Luc promoter and individual site mutations as
indicated. Constructs were transfected into HUVECs as described under
"Materials and Methods," and cultures were exposed to normoxia or
to hypoxia for 24 h. Luciferase expression is normalized to a
control plasmid, and activities are expressed relative to the aerobic
wild type. B, the wild type 176 ET-1 promoter or
individual mutations (4 µg each) were cotransfected into HUVECs with
AP-1, GATA-2, or HIF-1 expression plasmids (2 µg each) as
indicated. Controls were cotransfected with 2 µg of empty vector.
Results are from at least four separate experiments in duplicate.
Light bars, aerobic; dark bars, hypoxic.
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It was reported previously that the decreased basal expression of AP-1
and GATA-2 site mutations in the ET-1 promoter were complemented by
overexpressing the corresponding factor (23). To test for
complementation of the response to hypoxia, wild type and mutated
promoter constructs were cotransfected with HIF-1
, c-Jun, or GATA-2
expression vectors. These results are shown in Fig. 1B.
Expression of the wild type promoter was augmented by c-Jun (1.3-fold)
and GATA-2 (2.3-fold) cotransfections but not by HIF-1
, (all
p < 0.02; n = 4). The fold activation
of the wild type promoter by hypoxia was not significantly effected in
either case. Expression of the HIF-1 site mutation was not
significantly affected by cotransfection of c-Jun, GATA-2, or HIF-1
,
and there was no hypoxia-mediated induction of this construct under any condition. The basal expression of the AP-1 and GATA-2 site mutations was fully complemented by either c-Jun or GATA-2 cotransfection, in
agreement with a previous report (23), and the response to hypoxia was
fully restored to both mutations by cotransfection of c-Jun, GATA-2, or
HIF-1
. Cotransfection of cDNA encoding Sp1 did not restore the
hypoxia response of these mutations (data not shown).
These results demonstrate that the HIF-1
binding site is essential
but not sufficient for activation of the ET-1 promoter by hypoxia.
Homologous complementation of the AP-1 and GATA-2 site mutations and
cross-complementation by the heterologous factors suggest that each of
these factors can be recruited to the HIF-1 complex without directly
binding DNA. Complementation of either AP-1 or GATA-2 site mutations by
HIF-1
further suggests that these sites may modulate the DNA binding
affinity of HIF-1
(see "Discussion").
Gel Electrophoretic Mobility Shift Assays--
Gel mobility shift
assays were carried out to determine whether hypoxia changed the
binding activities of the HIF-1 site flanking proteins. As shown in
Fig. 2, shifts corresponding to the
binding of AP-1, GATA-2, and HIF-1 complexes were observed, confirming previous reports from this and other laboratories (23, 28). The
arrows in the upper panels indicate the positions
of specific AP-1-, GATA-2-, and HIF-1-shifted bands, respectively.
Using individual site sequences or the whole ET-1 HRE as the probe,
hypoxia did not effect the apparent abundance or binding activity of
either AP-1 or GATA-2. This is in contrast to some other cell types
where AP-1 binding is strongly activated by hypoxia (36). In the
top right panel, extracts from normoxic or hypoxic EC or
HeLa cells were reacted with the ET-1 HIF-1 probe (first
through fourth and seventh and eighth
lanes) or with a probe containing the erythropoietin (Epo)-HIF-1
consensus (fifth, sixth, and
ninth lanes). The ET-1-specific HIF-1 probe generated
markedly weaker interactions than the corresponding Epo-HIF-1 probe
(compare second, fourth, and sixth
lanes), suggesting that the ET-1 site is a low affinity HIF-1
binding site.

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Fig. 2.
Electrophoretic gel mobility shift analyses
of protein binding to the ET-1 HRE. Top panels, labeled
oligonucleotide probes containing the sequences corresponding to AP-1,
GATA-2, and HIF-1, wild type or mutant from the ET-1 HRE were mixed
with nuclear extracts from HUVECs cultured under normoxia or for
24 h under hypoxia as described under "Materials and Methods."
In the right panel, fifth, sixth, and
ninth lanes, the probe was the HIF-1 binding
sequence from the Epo HRE; HeLa cell nuclear extracts were used in the
third through sixth and ninth lanes.
Where indicated, competitor (unlabeled) oligonucleotide was added to
the binding reaction at 200-fold excess over the labeled probe.
Arrows indicate the specific band corresponding to the
indicated transcription factor. Bottom panels, labeled probe
was the complete ET-1 HRE, base pairs 91 to 142 (wild type) or with
the GATA-2 site mutated on the right. Where indicated,
specific antibodies (4-8 µl) were added to the binding reaction.
Arrows on the left of the panels
indicate factor-specific shifts; arrows on the
right indicate the positions of supershifts.
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Binding studies with a probe containing the complete ET-1 HRE-region
(
91 to
141) are shown in the lower panels. Shifts
representing AP-1 and HIF-1 were confirmed by supershifts with specific
antibodies (left panel, fourth and
sixth lanes). A weak supershift was observed with an
antibody directed against p300/CBP (fifth lane), confirming the presence of p300 in these complexes. The third arrow on
the left indicates the probable position of the GATA-2 band
shift; this band was always weak, and the anti-GATA-2 antibody did not generate a supershift (not shown). Mutation of the GATA-2 site in the
ET-1-HRE probe (lower right panel) did not affect the
binding of AP-1 or HIF-1 factors. Therefore, a functionally disruptive mutation (that prevented the promoter activation by hypoxia) did not
disrupt HIF-1 binding in vitro. Interestingly, the
supershift caused by the anti-c-Jun antibody also eliminated the
HIF-1-specific band (both bottom panels). These results show
that the ET-1 HRE binds the HIF-1 complex weakly, the binding of AP-1
GATA-2 is not affected by hypoxia, and there is no evidence for
cooperative binding in vitro.
Detection of Protein-Protein Interactions: Pull-down Assays--
A
biotinylated DNA pull-down assay was used to analyze protein-protein
interactions in the ET-1 HRE complex. This technique was used
previously by Ebert and Bunn to demonstrate cooperativity in the
transcriptional assembly of HIF-1, adjacent transcription factors, and
p300/CBP in the regulation of the LDH-A and Epo genes (37). In our
studies, biotinylated ET-1 HRE oligonucleotides with single, double, or
triple mutations were used to pull down proteins translated in
vitro as described under "Materials and Methods." Fig.
3A shows the results obtained
using a biotinylated ET-1 HRE with all transcription factor binding
sites intact or with all mutated. The small double arrows in
Fig. 3A indicate positions of HIF-1
(upper)
and HIF-1
(lower) products; in subsequent assays both
HIF-1 products were included in the reactions, but only the HIF-1
product was labeled with [35S]methionine during
translation. Interactions with the wild type probe were observed when
the factors were added individually or in combination. As expected, no
interactions were observed using the triple mutated oligonucleotide
(Fig. 3A, fifth lane).

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Fig. 3.
Pull-down analyses of protein binding to the
ET-1 HRE. Biotinylated oligonucleotide probes containing the wild
type ET-1 HRE sequence, and the single, double, or triple mutations
indicated were attached to magnetic beads and mixed with reticulocyte
lysates containing the indicated translated proteins. Bound proteins
were analyzed by gel electrophoresis as described under "Materials
and Methods." In panel A, proteins bound to the wild type
or triple mutation probe are shown; panel B shows single
mutations, and panel C compares a single mutation with a
double AP-1/GATA-2 mutation. Panels D and E show
proteins bound to double AP-1/GATA-2 and single HIF-1a site mutations,
respectively. In all cases arrows indicate specific factors;
the small double arrows in panel A indicate the
positions of HIF-1 (upper) and HIF-1 (lower). Both proteins are labeled with 35S in
panel A; in the other panels HIF-1 was always
present with HIF-1 but was not labeled. Results are representative
of three experiments.
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Individual (single) mutations of the AP-1 or GATA-2 sites in the ET-1
probe did not prevent the binding of c-Jun, GATA-2, or HIF-1 proteins
(Fig. 3B). This seemingly anomalous result can be explained
by cross-interactions between c-Jun and GATA-2 proteins. Previous work
has established that these proteins interact in vitro, and
there are high endogenous levels of both factors in rabbit reticulocyte
lysates (23 and data not shown). Therefore, single mutations of the
AP-1 or GATA-2 binding sites did not prevent the pull down because
protein bound to the remaining intact site is sufficient to pull down
both proteins. In support of this, double mutation of AP-1 and GATA-2
sites dramatically reduced the binding of both factors (Fig.
3C).
The pull down of GATA-2 and c-Jun by the double mutation probe was
enhanced when HIF-1 (
and
) was included in the binding reaction
(Fig. 3D), indicating that more GATA-2 and c-Jun complexed in the presence of HIF-1. It was possible to detect this interaction because the reticulocyte lysate, present in all binding reactions, does
not contain detectable endogenous HIF-1-
(data not shown). In
further support of this binding activity, when the HIF-1 site was
mutated, HIF-1 binding was reduced dramatically (Fig. 3E) but was recovered when additional GATA-2 or c-Jun was added. The latter
effect was sometimes masked by the high background level of HIF-1
binding even to the HIF-1-mutated probe. This background can also be
attributed to cross-interactions with AP-1, GATA-2, or p300 from the
lysate (see below). Importantly neither HIF-1 nor any of the other
factors bound to the triple mutation probe, indicating that all
interactions observed were dependent on specific binding sites. These
results provide strong support for physical interactions (direct or
indirect) among c-Jun, GATA-2, and HIF-1 bound to the ET-1 HRE. We were
unable to demon-strate similar interactions in
coimmunoprecipitation experiments using antibodies against
c-Jun, GATA-2, or HIF-1 (data not shown). This suggests that the
interactions require a DNA template and implicates physical associations with other factors such as p300 which may mediate complex
formation and cross-interactions of the factors (see below).
Function of the ET-1 HRE Complex: Role of p300--
The studies
described in Figs. 1-3 indicate that AP-1, GATA-2, HIF-1 (and NF-1)
proteins interact directly or indirectly when bound to DNA to produce a
functional response to hypoxia. The activator/adaptor protein p300/CBP
has been show to interact with AP-1, GATA factors, and HIF-1
(37-41). Therefore one function of AP-1 and GATA-2 here may be to
facilitate the recruitment of p300 to the ET-1 HRE. To test for this
constructs containing the wild type ET-1 HRE or different mutations
were cotransfected into endothelial cells with a p300 expression vector
(Fig. 4A). Cotransfection of
p300 augmented the basal and induced expression of the wild type ET-1
promoter but did not change the fold induction by hypoxia (2.01-fold
with p300 compared with 2.43-fold without p300; not significant,
n = 5). Therefore, p300 availability may limit both basal and activated expression of the ET-1 promoter in endothelial cells. Cotransfection of p300 also augmented the expression of the
HIF-1 site mutation but did not support induction by hypoxia; in fact,
expression of the HIF-1 site mutation was significantly less under
hypoxia under these conditions (p < 0.05). p300
overexpression augmented the basal expression of AP-1 and GATA-2 site
mutations by 2-3-fold, and remarkably, the hypoxia response was fully
restored to both promoters (p < 0.01;
n = 4 for both mutations). Therefore, the hypoxic
induction of AP-1 and GATA-2 site mutations can be complemented by
AP-1, GATA-2, HIF-1
, or p300.

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Fig. 4.
Regulation of ET-1 promoter expression by
p300/CBP. HUVECs were transfected with the wild type ET-1
176-Luc construct or individual mutations in the HRE as described in
Fig. 1. Panel A, the different constructs were cotransfected
with a vector expressing p300, and expression of reporter was measured
after normoxic or hypoxic culture as indicated. Panel B, the
wild type ET-1-Luc plasmid was cotransfected into HUVECs with a plasmid
expressing the adenovirus E1A protein with or without p300. In this
panel, right side, a construct containing
four copies of the Epo HRE was cotransfected with or without pE1A into
HeLa cells. In panel C, confluent HUVECs were infected with
adenovirus YH47928 (5 Pfu/cell) expressing E1A (32) for
24 h and were exposed to hypoxia for an additional 24 h or
remained aerobic. RNA was extracted and analyzed by Northern blots as
described previously using ET-1 or LDH-M cDNA probes (19,
48).
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The restoration of function by p300 suggests that at least one function
of AP-1 and GATA-2 is the recruitment of p300 to the ET-1 HRE complex.
This being the case, depletion of p300 should reduce the response as it
does with the LDH-A promoter and Epo 3'-enhancer sites (37). To test
for this, p300 availability was reduced by cotransfecting the p300
binding site of adenovirus E1A (Fig. 4B). Expression of the
wild type ET-1 promoter was quenched in the presence of the E1A
plasmid, but again the fold induction by hypoxia did not change
(2.3-fold without E1A; 2.2-fold with E1A; not significant,
n = 6). Cotransfection of E1A also quenched the
amplified expression caused by p300 overexpression, but yet again did
not change the fold induction. In contrast to this, E1A cotransfection
reduced the hypoxic fold induction but not the basal expression of the
Epo-HRE in HeLa cells (Fig. 4B). We also measured the
effects of E1A expression on the endogenous ET-1 transcript. As shown
in Fig. 4C, ET-1 transcripts were reduced by >90% in ECs
infected with adenovirus, but the message was still induced by hypoxia.
Both the basal expression and hypoxic induction of LDH were lost, and
there was no change of ribosomal 28 S RNA. Therefore p300 appears to
modulate the basal expression of the ET-1 gene promoter but not the
fold induction by hypoxia. These results suggest that p300 may modify
the level of expression of the ET-1 HRE possibly through interactions
with AP-1 and GATA-2, but the hypoxia response is independent of this regulation.
To determine whether hypoxia activation correlated with p300 binding,
biotinylated probes were used to pull down p300 from normoxic and
hypoxic nuclear extracts. As shown in Fig.
5, p300 bound equally to the wild type
probe and to all probes with single site mutations. There was no
difference in p300 binding between normoxic and hypoxic nuclear
extracts, and this pattern did not change with higher stringency washes
(not shown). p300 binding to the AP-1/GATA-2 double mutation probe was
reduced dramatically, but again there was no apparent difference
between normoxia and hypoxia, suggesting that the ET-1 HIF-1 site is
only a weak binding site for p300 compared with AP-1 or GATA-2. There
was no detectable p300 bound to the triple mutation probe as expected,
and binding to the phosphoglycerate kinase-HRE probe was highly
hypoxia-dependent, confirming that p300 binds to HIF-1 with
this probe.

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Fig. 5.
p300 binding to the ET-1 HRE. Nuclear
extracts were prepared from HUVECs grown under normoxic or (24 h)
hypoxic conditions. Biotinylated probes containing wild type or mutated
ET-1 oligonucleotides were mixed with the extracts, washed, and
analyzed by Western blot as described under "Materials and
Methods." The bottom panel on the right shows
p300 binding to a biotinylated oligonucleotide containing three copies
of the phosphoglycerate kinase (PGK) HRE (49) by the same
assay. Results are representative of three separate experiments.
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The Flanking Sites Are Not Strictly
Position-dependent--
Taken together, these results
suggest that the ET-1 HRE flanking sites may stabilize HIF-1
binding
to DNA and the interaction with p300. Because the GATA-2, HIF-1, and
AP-1 sites are closely aligned in the ET-1 promoter, we sought to
determine whether their functions were position-dependent.
50 base pair spacers were inserted between GATA-2 and HIF-1, and HIF-1
and AP-1 sites respectively, and the function of the HRE was
determined. As shown in Fig. 6, insertion of the spacers reduced basal expression of the promoters but
did not affect the fold induction by hypoxia.

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Fig. 6.
Position dependence of the ET-1, AP-1, and
GATA-2 flanking sequences. 50 base pairs of random DNA sequence
were inserted in between AP-1 and HIF-1, and HIF-1 and GATA-2 sites as
indicated. The expression and regulation of the promoters after
transfection into HUVECs were as described in Fig. 1.
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Induction of Et-1 Expression by Hypoxia Is Endothelial
Cell-specific--
We reported previously that the induction of the
endogenous ET-1 transcript by hypoxia was confined to endothelial cells
with no apparent activation in HeLa cells or cardiac myocytes (19). To
see if this also applied to the regulation of the promoter, the wild
type ET-1 construct was analyzed in HeLa, HepG2, and HEK-293 cells. As
shown in Fig. 7A, none of
these cell lines supported hypoxic induction of this promoter
(p < 0.001). Insertion of the
-enolase HRE, a
non-tissue-selective HRE, into the ET-1 promoter eliminated the tissue
selectivity, indicating that the selectivity was the property of the
ET-1 HRE rather than other promoter elements (Fig. 7B). None
of the recognized ET-1 HRE-binding factors is strictly EC-specific,
although GATA-2 has been shown to contribute to EC-specific gene
expression (42, 43). Therefore, we analyzed the effect of
cotransfecting other factors into HEK-293 cells. As shown in Fig.
7C, the hypoxia response was fully reconstituted by
cotransfecting GATA-2 and HIF-1
but not by p300 or the other plasmid
combinations. This supports the essential role of GATA-2 (and AP-1)
factors in creating an active HRE complex and accounts at least in part
for the apparent tissue specificity of this response.

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Fig. 7.
Tissue-specific regulation of the ET-1
HRE. In panel A the wild type ET-1 176 promoter was
transfected into HeLa, HepG2, or HEK-293 cells as indicated, and
expression was measured after exposure to normoxic or hypoxic
conditions. The control plasmid, pGL3 (Promega), containing three
copies of the Epo HRE was induced 8.7-fold (± 0.5, n = 6) in HeLa cells. Panel B, expression of the ET-1 promoter
containing one or two copies of an enolase-1 HRE transfected into HeLa
cells. Panel C, effects of cotransfecting AP-1, GATA-2,
HIF-1 , and p300 vectors with the wild type ET-1 promoter in HEK-293
cells.
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DISCUSSION |
In this report we have shown that individual mutations of the
GATA-2, HIF-1, AP-1, or NF-1 sites in the ET-1 proximal promoter eliminated activation of the promoter by hypoxia. The HIF-1 site mutation eliminated the hypoxia response under all conditions whereas
AP-1 and GATA-2 site mutations were fully complemented by
overexpressing AP-1, GATA-2, HIF-1, or p300/CBP factors. These results
implicate functionally important protein-protein interactions between
these factors that do not necessarily require the direct DNA binding of
all factors. In addition, this is the first evidence of roles for GATA
factors or NF-1 in the transcriptional activation of a promoter by
hypoxia. Although we have not addressed protein binding to the NF-1
site in this report, mutation of the site eliminated hypoxia-induction
and it seems possible that the ubiquitous NF-1 binding proteins
function in a manner similar to AP-1 and GATA-2 proteins in the HRE complex.
Pull-down binding assays of in vitro translated proteins
demonstrated that c-Jun and GATA-2 proteins associated with the ET-1 HRE probe even when their individual sites were mutated, supporting the
presence of strong protein-protein interactions between these factors
and the HRE complex (22, 23). The pull-down of these factors was
dramatically decreased when the probe contained a double AP-1/GATA-2
mutation, confirming the requirement for at least one DNA binding site.
The enhanced pull down of AP-1 and GATA-2 by the double mutation probe
in the presence of added HIF-1 factors indicates that these factors can
be pulled down by interacting directly with HIF-1 or other factors
associated with DNA-bound HIF-1. These results confirm the physical as
well as functional relationships among HIF-1, AP-1, and GATA-2 factors.
It is noteworthy that we did not observe strong protein-protein
interactions in DNA-free coimmunoprecipitation assays of these factors
under conditions where the expected interactions between c-Fos and
c-Jun were seen (data not shown). This result supports the requirement
of DNA binding for assembly of the complex and subsequent
cross-interaction among individual factors.
Hypoxic activation was restored to ET-1 promoters with GATA-2 or AP-1
mutations by overexpressing the homologous factors or by overexpressing
either HIF-1
or p300. Complementation of AP-1 and GATA-2 site
mutations with p300 demonstrates the central role of p300 in organizing
the cooperative binding of these transcription factors to the ET-1 HRE
site. This confirms previous work that demonstrated a similar role for
p300/CBP in the cooperative binding of HIF-1 flanking site proteins to
the LDH-A promoter and erythropoietin 3'-enhancer (37). In these latter
studies mutation of the flanking cyclic AMP response element
site in the LDH-A HRE reduced p300 recruitment and prevented activation
by hypoxia. Complementation of the ET-1 HRE flanking site mutations by
HIF-1
overexpression in our studies suggests that these mutations
also reduce the affinity of HIF-1 for DNA binding, an effect that may
be independent of p300. These studies support dual roles for the ET-1
HIF-1 flanking sites, modulating the affinity of HIF-1 binding and
promoting recruitment of p300.
The ET-1 HRE differs markedly from the LDH-A and Epo HREs in having two
and perhaps three strong p300 binding sites in addition to the HIF-1
complex. Whereas p300 binding to the LDH-A and Epo HRE sites is
strictly dependent on hypoxia ( Ref 37; also see Fig. 4C),
this is not the case for the ET-1 HRE site. p300 bound strongly to the
wild type ET-1 HRE and to all single site mutations but only weakly to
the double AP-1/GATA-2 mutation, and there was no evidence for
increased p300 binding from hypoxic nuclear extracts. Importantly, this
indicates that p300 binds principally to the ET-1 AP-1 and GATA-2 sites
in a constitutive manner. This is in contrast to the LDH-A and Epo HRE
sites and may account for important differences in the functions of
these HREs. In particular, the absence of hypoxia-regulated p300
binding to the ET-1 probes is consistent with a similar absence of p300
influence on the fold activation of the ET-1 promoter by hypoxia (Fig.
4, A and B). This again is in contrast to the
LDH-A promoters and Epo 3'-enhancer where fold activation correlated
quantitatively with p300 availability and binding (37, 41). Therefore,
p300 binding controls the fold activation of the LDH-A and Epo genes by
hypoxia but not the ET-1 promoter. Our data are consistent with a model
whereby the HIF-1 flanking sites of the ET-1 HRE determine the affinity of both HIF-1 and p300 binding. It is also possible that the wild type
ET-1 HRE has a high affinity p300 binding site that is saturated even
under conditions of p300 depletion; in this case it would be predicted
that this high affinity site is lost when any of the flanking sites is
mutated. We have no direct evidence for the latter possibility.
The modulation of ET-1 HRE function by the flanking sites also appears
to dictate EC selectivity of the response. In contrast to other
HRE-dependent promoters, the wild type ET-1 promoter was
not responsive to hypoxia in HeLa, HepG2, or HEK-293 cells (19). The
response could be reconstituted by overexpressing GATA-2 and HIF-1
.
This result underscores the importance of these factors in regulating
the response of the ET-1 HRE to hypoxia and supports a mechanism
whereby flanking site factors modulate of HIF-1 binding affinity
through cell-specific protein-protein interactions.
Previous studies have implicated essential contributions of AP-1 to the
hypoxia-mediated activation of tyrosine hydroxylase, heme oxygenase,
and possibly vascular epithelial growth factor promoters (36, 44-47).
It seems possible that AP-1 may fulfill similar roles in mediating
HIF-1 and p300 binding activities also in these promoters. In each case
the AP-1 site is situated further from the HIF-1 binding site than is
the case with the ET-1 promoter; however, our results suggest that
immediate proximity to the HIF-1 site may not be a critical
parameter for the modulating roles of these factors. The ET-1
promoter may be unique in requiring not only AP-1 but also GATA-2 and
NF-1 factors for a functional HRE. One important property dictated by
this multiple site regulation, which conveys a degree of tissue
selectivity, may be to ensure that this potent vasoconstrictor is not
activated adventitiously by hypoxia in tissues other than the endothelium.