Contributions of HIF-1, AP-1, GATA-2, and CBP/p300

Molecular Regulation of the Endothelin-1 Gene by Hypoxia: Contributions of HIF-1,

AP-1, GATA-2, and CBP/p300

Kazuhito Yamashita, Daryl J. Discher, Jing Hu, Nanette H. Bishopric, and Keith A. Webster†.

Department of Molecular and Cellular Pharmacology, University of Miami Medical Center,

Miami, FL. 33149

Key Words: gene regulation, ET-1, ischemia, endothelium,

Running Title:Regulation of ET-1 by Hypoxia

†Address for Correspondence

Keith A. Webster

Department of Molecular and Cellular Pharmacology

Rosenstiel Medical Science Building, Rm. 6038

University of Miami Medical Center,

1600 NW 10th Avenue, Miami, FL 33136

Tel. (305) 243-6779. Fax. (305) 243-3082.

Email. [email protected]

ABSTRACT

Endothelin-1 (ET-1) is a peptide hormone with potent vasoconstrictor properties that is

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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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synthesized and secreted predominantly by vascular endothelial cells. Its production is regulated

by numerous stimuli including ischemia and hypoxia, and the enhanced levels that occur during

myocardial ischemia may contribute to the progression of heart failure. We previously reported

a preliminary characterization of a hypoxia-inducible factor-1 (HIF-1)-binding site in the

human ET-1 promoter that contributed to the activation of ET-1 expression in endothelial cells.

We report here that the HIF-1 binding site alone is not sufficient for the response to hypoxia but

requires an additional 50 bp of flanking sequence that include binding sites for the factors AP-1,

GATA-2 and CAAT-binding factor (NF-1). Mutation of any one of these sites or the HIF-1

site eliminated induction by hypoxia. Mutations of the AP-1 and GATA-2 sites, but not the

HIF-1 site were complemented by over-expressing AP-1, GATA-2, HIF-1α or the activator

protein p300/CBP, restoring the response to hypoxia. Binding studies in vitro confirmed

physical associations between GATA-2, AP-1, and HIF-1 factors. Over-expression or

depletion of p300/CBP modulated the level of ET-1 promoter expression as well as the

endogenous ET-1 transcript, but did not change the fold induction by hypoxia in either case.

Regulation of the ET-1 promoter by hypoxia in non-endothelial cells required over-expression

of GATA-2 and HIF-1α. The results support essential roles for AP-1, GATA-2, and NF-1, in

stabilizing the binding of HIF-1 and promoting recruitment of p300/CBP to the ET-1 hypoxia

response complex.

INTRODUCTION

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Endothelin-1 (ET-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 (reviewed in (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 (reviewed in

(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), TGF-β (8), shear stress (9,10), TNF-α

(11), interleukin-1 (12), insulin (13), angiotensin II (14), nitric oxide (15), and hypoxia (15-20). Molecular anal

gene have revealed cis-acting sequences that contribute to ET-1 expression (21). The promoter

region contains AP-1, GATA-2, 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 that probably determine the short (15-20 min.) half-life

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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 NO and CO and mimicked by transition metals (15-17). The ET-1

promoter contains an inverted HIF-1 binding site at position -118 bp upstream of the

transcription start site that binds the factors HIF-1α and ARNT (HIF-1β) and is essential for the

promoter response to hypoxia (20,28). Here we report that this response also requires three adjacent

transcription factor binding site to form a functional hypoxia-responsive complex. The response

is EC-cell specific and is modulated but not necessarily dependent on interactions with the

activator protein p300/CBP.

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 (PBS), and filled with 0.1% collagenase in PBS

containing Ca2+ and Mg2+. After incubation at 37oC 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 penicillin (100 U/ml),

streptomycin (100 µg/ml), L-glutamine, and 10% human serum. In some experiments HUVECs

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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,

Philidelphia, PA). 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 PCR-based procedures. The

primers were GATA-2: 5’CCGACTCCGGCTGCACGTTGCCTGTTGGTGACTAATA AC3’;

AP-1: 5’GACACCTAATAACACAATAACATTGTCTGGGGCTGG3’; with the appropriate

opposite strand primers, and NF1: 5’AACAACATTGTCTGGGGCTGGAAT3’ +

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,

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pEnoHRE, and adenoviral vectors expressing E1A have been described previously (32).

Plasmid Insertions. A 70 bp fragment from the enolase-1 promoter containing the enolase-1

HRE (33) was inserted into the Bam H-1 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 PCR-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’ ATGCTAGGCGTCATGAGTACG

AGGTCGGAGCTACGTACTGCCGTTGTACG. 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 β-actin (29) were labeled by random priming

(Prime-It kit, Stratagene, CA) 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 β-actin

as the control.

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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 24-48h. 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; ET-1HIF-1(WT) 5’ CTCCGGCTGCAC

GTTGCCTG; ET-1HIF-1(M) CTCCGGCTTACCGTTGCCTG; AP-1 (WT) GCCTGTTGG TG

ACTAATAAC; AP-1(M), GCCTGTTGACACCTAATAAC; GATA-2 (WT) 5’ CCTGGCTTA

TCTCCGGCTGC; GATA-2 (M) 5’ GGCCTGGCCCGACTCCGGCT; ET-1HRE(WT), 5’ GG

CCTGGCTTATCTCCGGCTGCACGTTGCCTGTGGGTGACTAATCACA. Site mutations

within the ET-1HREWT 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 [γ-32P] ATP (NEN/DuPont). Equal amounts of

radioactive probe (1.5 - 2.5 x 104 cpm) were added to binding reactions that contained 8 µg of

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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) (Pharmacia). Antibodies

against c-Jun, GATA-2, Sp1, c-Fos, and p300 were from Santa Cruz Biotechnology; anti-HIF-

1α antibody was from Lab Vision Corp. (Freemont, CA). Reactions were incubated for 15 min.

at 22oC before separating on non-denaturing 6% polyacrylamide gels at 4oC (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,

Tennessee) GATA-1 and GATA-2, (generous gifts from S. Orkin, Dana Farber Cancer Institute,

Harvard Medical School, Boston MA) HIF-1α (generous gift from F. Bunn, Brigham and

Women’s Hospital, Harvard Medical School, Boston MA) HIF-1β (ARNT) (generous gift from

Darren Richard, Institute of Signaling, Developmental Biology and Cancer Research, CNRS,

Nice Cedux 2, France). All cDNAs were subcloned into pBlueSkript SK (-) by standard cloning

procedures. The corresponding proteins were transcribed and translated in vitro using rabbit

reticulocyte lysates (Promega Corporation).

Pull-Down Assays Double stranded probes biotinylated at the 5’ ends were from BRL.

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

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Streptavidin (Dynal, Inc., Lake Success, NY) were prepared by washing three times in phosphate

buffered saline pH 7.4 containing 0.1% BSA and two times with Tris-EDTA containing 1M

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 21oC. Equal cpms of proteins translated in vitro were made to 1X

with binding buffer and mixed with approximately 100 µg of Dynabeads containing 10 p.mol of

the individual oligonucleotide probe in a final volume of 250µl. The mixture was rotated at room

temperature for 20 min and proteins bound to the beads were separated from unbound proteins

by successive washes; 3 times with 0.5X binding buffer, and once with 1X binding buffer.

Higher stringency wash included two washes with 2X binding buffer. Beads and bound proteins

were pulled down with a magnetic concentrator, suspended in 1X sample buffer, boiled for 5 min

and resolved on 10% polyacrylamide-SDS gels 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 electro-blotted onto nitrocellulose membranes (BioRad).

Blots were stained with Ponceau Red to monitor the transfer of proteins. Membranes were

blocked for 1 hour at room temperature with 5% non-fat 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 hours in the same buffer. After washing, the blots were incubated for 1 hr with 1:7500 dilution

of horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or HRP-conjugated donkey anti-

goat IgG and visualized using enhanced chemiluminescence (Pierce).

RESULTS

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Functional Characterization of the ET-1 Promoter HRE.

Figure 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 in Methods. The expression of the wild type promoter and each mutation in

endothelial cells cultured under aerobic or hypoxic conditions is shown in Figure 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.

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 over-expressing the corresponding

factor (23). To test for complementation of the response to hypoxia, wild type and mutated promoter

constructs were co-transfected with HIF-1α, c-Jun, or GATA-2 expression vectors. These

results are shown in Figure 1B. Expression of the wild type promoter was augmented by c-Jun

(1.3-fold) and GATA-2 (2.3-fold) co-transfections, but not by HIF-1α, (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 effected by co-transfection of

c-Jun, GATA-2, or HIF-1α, 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 co-transfection, in agreement with a previous report

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(23), and the response to hypoxia was fully restored to both mutations by co-transfection of c-Jun,

GATA-2, or HIF-1α. Co-transfection of cDNA encoding Sp1 did not restore the hypoxia

response of these mutations (data not shown).

These results demonstrate that the HIF-1α 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 suggests 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-1α further suggests that

these sites may modulate the DNA binding affinity of HIF-1α (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 Figure 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-hand panel, extracts from normoxic or

hypoxic EC or HeLa cells were reacted with the ET-1 HIF-1 probe (Lanes 1-4, 7 and 8), or with

a probe containing the erythropoietin (Epo) HIF-1α consensus (lanes 5,6, and 9). The ET-1-

specific HIF-1 probe generated markedly weaker interactions than the corresponding Epo-HIF-1

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probe (compare lanes 2,4 and 6) suggesting that the ET-1 site is a low-affinity HIF-1 binding

site.

Binding studies with a probe containing the complete ET-1 HRE-region (-91 to -141) is

shown in the lower panels. Shifts representing AP-1 and HIF-1 were confirmed by super-shifts

with specific antibodies (lanes 4 and 6). A weak supershift was observed with an antibody

directed against p300/CBP (lane 5), confirming the presence of p300 in these complexes. The

lower arrow 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 super-shift (not shown). Mutation of the

GATA-2 site in the ET-1-HRE probe (lower right panel) did not effect 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 and

GATA-2 is not effected 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 in Methods. Figure 3A shows the results obtained using a

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biotinylated ET-1 HRE with all transcription factor binding sites intact or with all mutated. The

small double arrows in figure 3A indicate positions of HIF-1α (upper) and HIF-1β (lower)

products; in subsequent assays both HIF-1 products were included in the reactions but only the

HIF-1 α 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 (Figure 3A lane

5).

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 (Figure 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

(Panel 3C).

The pull-down of GATA-2 and c-Jun by the double mutation probe was enhanced when

HIF-1 (α and β) were included in the binding reaction (Panel 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-α (data not shown). In further support of this binding activity, when the

HIF-1 site was mutated, HIF-1 binding was dramatically reduced (Figure 3E), but was

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recovered when additional GATA-2 or c-Jun were 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) between c-Jun,

GATA-2 and HIF-1 bound to the ET-1 HRE. We were unable to demonstrate similar

interactions in co-IP 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 that may mediate complex formation and cross-

interactions of the factors (see below).

Function of the Et-1 HRE Complex: The Role of p300

The studies described in Figures 1 through 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-1α (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 co-transfected into endothelial cells with a

p300 expression vector (Figure 4A). Co-transfection 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; NS, n = 5). Therefore, p300

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availability may limit both basal and activated expression of the ET-1 promoter in endothelial

cells. Co-transfection 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 over-expression

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-1α, or p300.

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 (Figure 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; NS; n=6). Co-transfection of E1A also quenched the amplified

expression caused by p300 over-expression, but yet again did not change the fold induction. In

contrast to this, E1a co-transfection reduced the hypoxic fold induction but not the basal

expression of the Epo-HRE in HeLa cells (Figure 4B). We also measured the effects of E1A

expression on the endogenous ET-1 transcript. As shown in Figure 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 28S RNA. Therefore p300 appears to modulate the basal expression of the

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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

Figure 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 dramatically reduced, 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.

The Flanking Sites are not Strictly Position-Dependent

Taken together, these results suggest that the ET-1 HRE flanking sites may stabilize

HIF-1α 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. Fifty 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 Figure 7, insertion of the spacers reduced basal expression of the promoters but did not affect

the fold induction by hypoxia.

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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 Figure 7A, none of these cell lines

supported hypoxic induction of this promoter (p< 0.001). Insertion of the β-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

(Figure 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 co-transfecting other factors into HEK-293 cells. As shown in Figure 7C,

the hypoxia response was fully reconstituted by co-transfecting GATA-2 and HIF-1α 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.

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 over-expressing AP-1, GATA-2,

HIF-1, or p300/CBP factors. These results implicate functionally important protein-protein

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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 between HIF-1, AP-1, and

GATA-2 factors. It is noteworthy that we did not observe strong protein-protein interactions in

DNA-free co-immunoprecipitation 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

between individual factors.

Hypoxic activation was restored to ET-1 promoters with GATA-2 or AP-1 mutations by

over-expressing the homologous factors, or by over-expressing either HIF-1α or p300.

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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 CRE 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-1α over-expression in our studies suggest 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 2 and

perhaps 3 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 ((37), also see Figure 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. (Figure 4A and B). This again is in contrast to the LDH-A

promoters and Epo 3’ enhancer where fold activation correlated quantitatively with p300

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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 are 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 over-expressing GATA-2 and HIF-1α. 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 VEGF 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

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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.

ACKNOWLEDGMENTS

This work was supported by grant # HL44578 from the National Institutes of Health (to K. A.

W.), and by a Postdoctoral Fellowship from the American Heart Association, Florida Affiliate,

(to KY).

A preliminary report of this work has been published in abstract form (Yamashita et al,

Circulation, 98: I 743, 1999).

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FIGURE LEGENDS

Figure 1. Functional regulation of the ET-1 HRE. Top indicates location and sequence of the

ET-1 HRE and positions of the transcription factor binding sites. Below the sequence base

changes for each site mutation are indicated. A. Relative luciferase activity of the wild type -

176 ET-1-luciferase promoter and individual site mutations as indicated. Constructs were

transfected into HUVECs as described in Methods, and cultures were exposed to normoxia or to

hypoxia for 24h. 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 co-transfected into HUVECs with AP-1, GATA-2 or HIF-1α

expression plasmids (2 µg each) as indicated. Controls were cotransfected with 2 µg of empty

vector. Results are from at least 4 separate experiments in duplicate. Light columns: aerobic;

dark columns: hypoxic.

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Figure 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 (WT) or mutant (M) from the ET-1 HRE were mixed with

nuclear extracts from HUVECs cultured under normoxia or for 24 h under hypoxia as described

in Methods. In the right hand panel, lanes 5, 6, and 9, the probe was the HIF-1 binding sequence

from the Epo HRE; HeLa cell nuclear extracts were used in lanes 3-6, and 9. 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 (wt) or with the GATA-2 site mutated on the right. Where indicated specific antibodies

(4-8µL) 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.

Figure 3. Pull-down analyses of protein binding to the ET-1 HRE. Biotinylated

oligonucleotide probes containing the wild type ET-1 HRE sequence, and 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 in methods. In panel A proteins bound to the wild type (WT) or

triple mutation (M) probe are shown; panel B show single mutation and panel C compares 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

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specific factors; the small double arrows in 3A indicate the positions of HIF-1 α (upper) and

HIF-1 β (lower), both proteins are labeled with 35S in panel A, in the other panels HIF-1β was

always present with HIF-1α, but was not labeled. Results are representative of 3 experiments.

Figure 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

Figure 1. In 4A. the different constructs were cotransfected with a vector expressing p300 and

expression of reporter was measured after normoxic or hypoxic culture as indicated. In 4B the

wild type ET-1-Luc plasmid was cotransfected into HUVECs with a plasmid expressing the

adenovirus E1A protein with or without p300. In 4B, right-hand side a construct containing 4

copies of the Epo HRE was cotransfected with pE1A into HeLa cells. In 4C confluent HUVECs

were infected with Adenovirus YH47928 (5 pfu/cell) expressing E1A (32) for 24 h, and were

exposed to hypoxia for an additional 24h or remained aerobic. RNA was extracted and analyzed

by Northern blots as described previously using ET-1 or LDH-M cDNA probes (19,48)

Figure 5. p300 binding to the ET-1 HRE. Nuclear extracts were prepared from HUVECs grown

under normoxic or (24h) 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 in Methods. The bottom right hand panel shows p300 binding to a biotinylated

oligonucleotide containing 3 copies of the phosphoglycerate kinase (pgk) HRE (49) by the same

assay. Results are representative of 3 separate experiments.

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Figure 6. Position dependence of the ET-1, AP-1, and GATA-2 flanking sequences. Fifty 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 was as described in Figure 1.

Figure 7. Tissue-specific regulation of the ET-1 HRE. In 7A 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 3 copies of the Epo HRE was induced by 8.7 fold (± 0.5, n=6) in HeLa

cells. B. Expression of the ET-1 promoter containing 1 or 2 copies of an enolase-1 HRE

transfected into HeLa cells. C. Effects of cotransfecting AP-1, GATA-2, HIF-1α, and p300

vectors with the wild type ET-1 promoter in HEK-293 cells

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-142 CGGGTCTTATCTCCGGCTGCACGTTGCCTGTGGGTGACTAATCACACAATAA -91

GATA-2 HIF-1 AP-1 CAAT TATAALuc

CCGA ACAC TACCTAC

0

1.0

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GATA-2 M

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1.0

2.0

3.0

4.0

Rel

ativ

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uci

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se A

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ity


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Normoxia

Hypoxic

Hypoxic + Comp

Hypoxic + c-Jun ab

ET1-GATA2M

Normoxia

Hypoxic

Hypoxic + Comp

Hypoxic + c-Jun ab

Hypoxic + HIF-1ab

Hypoxic + p300 ab

HIFAP1

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GATA-2

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oxia

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c-JunGATA

-2 + HIF-1

GATA-2 + c-Jun

c-Jun + HIF-1

c-Jun + HIF-1

GATA-2 + H

IF-1

GATA-2 + c-Jun

ET-1 AP-1-M ET-1 GATA-2-M

HIF-1 (α+β)

GATA-2

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ALL

ET-1 HRE -WT MProbe:

A. B.

GATA-2 + c-Jun

GATA-M

AP-1-M

Dble-MC.

c-Jun + HIF-1

GATA-2 + H

IF-1

GATA-2 + c-Jun

GATA/AP-1-MProbe:

c-Jun + HIF-1

GATA-2 + H

IF-1

HIF-1 (α+β)

HIF-1-M

D. E.

HIF-1 αβ

GATA-2c-Jun

HIF-1 GATA-2c-Jun


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WT

+ p300+E1A

HREEpo

E1A + p300+E1A

0

2.0

4.0

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8.0

10.0

12.0Aerobic

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B.

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AP-1 M GATA-2 MWT HIF-1 M

0

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+ p300

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A.R

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ET-1 AP-1 M

ET-1 GATA

-2 M

ET-1 HRE -W

T

ET-1 HIF-1 M

A HX A HXA HXA HX

p300

GATA-2/A

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A HXA HXA HX

ET-1 ALL M

PGK[HRE]3

p300


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GATA-2 HIF-1 AP-1 NF-1

TATAALuc

50 bp inserts

-176

0

1.0

2.0

3.0

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GATA-2//H

IF-1

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A HX A HXA HX

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0

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by guest on January 29, 2018 http://www.jbc.org/ Downloaded from

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Kazuhito Yamashita, Daryl J Discher, Jing Hu, Nanette H Bishopric and Keith A WebsterAP-1, GATA-2, and CBP/p300

Molecular regulation of the endothelin-1 gene by hypoxia: contributions of HIF-1,

published online January 22, 2001J. Biol. Chem.

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http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M011344200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2001/01/22/jbc.M011344200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2001/01/22/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2001/01/22/jbc.M011344200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

Documents

Contributions of HIF-1, AP-1, GATA-2, and CBP/p300