From the Zentrum für Molekulare Medizin der
Universität zu Köln, Joseph-Stelzmann-Strasse 9, 50931 Köln, Germany, § Abteilung Molekulare Zellbiologie,
Max-Planck-Institut für physiologische und klinische Forschung,
Parkstrasse 1, 61231 Bad Nauheim, Germany, ¶ Centre d'Immunologie
de Marseille-Luminy, INSERM-CNRS, Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France, and
Abteilung Neuropathologie,
Universität Erlangen-Nürnberg 91052, Nürnberg, Germany
Received for publication, November 5, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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Interactions between Ets family
members and a variety of other transcription factors serve important
functions during development and differentiation processes,
e.g. in the hematopoietic system. Here we show that the
endothelial basic helix-loop-helix PAS domain transcription
factor, hypoxia-inducible factor-2 The proliferation of vascular endothelial cells (which line the
inner surface of all blood vessels) is a key mechanism of vascular
growth involved in normal embryonic development and adolescence but is
also a prerequisite of tumor growth and the progression of other
destructive diseases (1, 2). Endothelial cell proliferation is mediated
primarily by vascular endothelial growth factor
(VEGF1; also known as VPF,
vascular permeability factor) signaling via its high affinity tyrosine
kinase receptors. The VEGF receptors are expressed prominently on
endothelial cells, thus establishing the cell specificity of VEGF. VEGF
signaling is also important for behavioral changes of the capillary
endothelium during blood vessel growth, such as invasive migration,
matrix degradation, and sprouting, and also for the survival of
endothelial cells in immature blood vessels. VEGF is induced in
response to hypoxia and/or low intracellular glucose, thus stimulating
neovascularization in accordance with the increasing metabolic demands
of a growing tissue (3-5). The function of the high affinity VEGF
receptors is not redundant. Although VEGF receptor-1 (Flt-1) is
necessary for early embryonic vascular integrity (6), VEGF receptor-2 (KDR in humans/Flk-1 in mouse) is indispensable for the formation of
the primordial vasculature during early embryogenesis; mouse embryos
with targeted disruption of the Flk-1 gene fail to
differentiate both blood and endothelial cells and die as a consequence
of avascularity (7). Consistent with this phenotype, Flk-1 is strongly
expressed by the mesodermal precursors of both endothelial and
hematopoietic cells (hemangioblasts) (8, 9). Later on, during ontogeny, Flk-1 is expressed primarily on proliferating endothelial cells but
becomes largely down-regulated in many vascular domains in the adult.
However, Flk-1 expression is reactivated in newly forming blood
vessels, e.g. in the corpus luteum, and in neoplastic
lesions that express high amounts of VEGF, such as glioblastomas (10, 11). The level of VEGF receptor expression appears to be influenced by
the availability of their ligand VEGF, which is secreted upon tissue vascularization (12, 13). Signaling in the embryonic endothelium
has been demonstrated convincingly only for Flk-1, whereas the role of
Flt-1 in the developing vasculature is still puzzling (14, 15).
Although other signaling systems, such as angiopoietins/tie2 receptor
and ephrins/eph receptors, as well are indispensable for vascular
differentiation (16-18), the role of Flk-1 signaling is pivotal
because inhibition of Flk-1 signaling, but not of any other receptor,
prevents vascular development from its beginning and consistently
inhibits tumor vascularization (2). Therefore, an understanding of
the mechanisms that regulate the expression of Flk-1 in the endothelium
would provide general insights into the mechanisms of vascular
development in health and disease and might open up new approaches to
the as yet frustrating therapy of ischemic and proliferative malignancies.
We recently characterized the essential regulatory elements of the
Flk-1 gene (19). A 939-bp 5'-flanking region together with a
430-bp minimal enhancer from the first intron was essential and
sufficient to convey endothelial cell-specific expression of the
lacZ reporter gene in the transgenic mouse. By mutational analysis in transgenic animals, distinct SCL/TAL-1, GATA, and Ets
consensus sites within the enhancer were shown to be essential for
Flk-1 gene expression (20), indicating that members of the SCL, GATA, and Ets transcription factor families are not only important
regulators of hematopoiesis (21) but also act upstream of Flk-1
expression during development. Consistently, members of all three
families are expressed by endothelial cells and have been implicated in
vascular development in the embryo and in tumor angiogenesis. SCL, a
candidate master regulator of hematopoiesis, appears also to be
involved in early endothelial cell differentiation and possibly acts
upstream of Flk-1 at least in zebrafish (22-24). Ets factors stimulate
the expression of a variety of endothelial cell-specific genes that are
involved in vascular growth and sprouting, including urokinase,
metalloproteinases, flt-1, tie-1, and tie-2 (25-30). Ets-1 is highly expressed in the lateral mesoderm when Flk-1
starts to be expressed in vascular endothelial cell precursors (31). In
addition, the up-regulation of Ets-1 in capillary-invading tumors can be correlated with re-expression of VEGF receptors (32). Also the Ets family members NERF2 and TEL appear to serve important functions during angiogenesis (29, 30, 33). Others are
important for cell lineage determination of lymphoid and monomyeloid cells such as PU.1 (34) or may play a critical role in the
differentiation of epithelia, such as ESE-1 and -2 (35).
In a variety of Ets-responsive promoters and enhancers analyzed thus
far, DNA binding and transactivation by Ets family transcription factors is enhanced by the formation of a complex with distinct members
of the bZIP and bHLH families of transcription factors (36). Recently,
we and others cloned a novel member of the bHLH PAS family of
transcription factors, which is expressed at high levels in vascular
endothelium in mouse and quail embryos but also in tissues expressing
high amounts of VEGF, and was denominated EPAS-1/HRF/HLF/MOP2 (37-41).
This factor is highly homologous to hypoxia-inducible factor-1 The expression pattern and the biological activity of HIF-2 Antibody Preparation, Immunohistochemistry, Western Blot
Analysis, and in Situ Hybridization--
A 2315-bp SacI
fragment comprising a large portion of the open reading frame of the
murine HIF-2 Cell Culture and Reagents--
HEK-293 cells were cultured in
DMEM-F12 (Invitrogen) supplemented with 10% fetal calf serum
(PAA Laboratories), 1 mM sodium pyruvate (Abimed),
nonessential amino acids (1%, Abimed), 0.4% (v/v)
Transient Expression Assays--
Plasmid DNAs were purified
using Qiagen columns. HepG2 and BAE cells were transfected by
electroporation. All other cell lines were transfected using
SuperfectTM transfection reagent (Qiagen). In a typical transfection
experiment 200 ng of reporter plasmid together with 200 ng of
expression vector and 2 ng of pRL-TK vector (Promega) were mixed in 20 µl of DMEM and SuperfectTM transfection reagent (Qiagen), and after
10 min at room temperature the mixture was added to freshly
passaged subconfluent HEK-293 cells in 24-well plates. 48 h after
transfection, cells were harvested in lysis buffer (Dual-LuciferaseTM
Reporter Assay System, Promega) and subsequently assayed for firefly
and Renilla luciferase in a Micro-Lumat-PlusTM (Berthold)
luminometer after the addition of luciferase assay reagent and
Stop & Glow substrates (Dual-LuciferaseTM Reporter Assay System,
Promega), respectively. To correct for variable transfection
efficiencies, the ratio of both luciferase activities was determined.
At least three independent transfection assays were performed. With the
exception of HepG2 and BAE cells, which were transfected by
electroporation, all other cells were treated in a similar manner with
only minor modifications.
Expression Vector Constructs and Mutagenesis--
The cloning of
HIF-1 Plasmid Constructs for Reporter Assays and Transgene
Analysis--
Isolation of the 5'-flanking region of the mouse
Flk-1 gene and preparation of the reporter construct have
been described previously (47). A 946-bp PCR fragment containing the
Flk-1 sequence from
To generate transgene constructs, PCR-based mutagenesis techniques were
applied to mutate the HIF-2 Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays--
Nuclear extracts from BAE cells were prepared at
4 °C essentially as described (48). Briefly, cells were scraped from
plates in phosphate-buffered saline, washed in 5 packed cell volumes of
hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT), resuspended in 3 packed cell volumes of hypotonic
buffer, and incubated on ice for 10 min. Nuclei were resuspended,
collected by centrifugation, and again resuspended in an 0.5 packed nuclear volume of low salt buffer (20 mM HEPES, pH
7.9, 25% glycerol, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF,
0.5 mM DTT). Under constant stirring, an 0.5 packed nuclear volume of high salt buffer (20 mM HEPES, pH7.9, 25%
glycerol, 1.5 mM MgCl2, 1.2 M KCl,
0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM
DTT) was added and mixed for 30 min under constant shaking. After
pelleting of cell debris, nuclear extract was dialyzed against dialysis buffer (20 mM HEPES, pH 7.9, 25% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF,
0.5 mM DTT). Aliquots of nuclear extracts were shock-frozen
in liquid nitrogen and stored at
An electrophoretic mobility shift assay was performed as described (48,
49) using 15 µg of nuclear extract and 10,000 cpm of labeled
oligonucleotide in a 10-µl reaction volume at 4 °C.
Oligonucleotides were annealed with their corresponding reverse complementary strands and radiolabeled using T4 polynucleotide kinase
and [ DNase I Footprint Analysis--
A 226-bp Flk-1 promoter fragment
was amplified using PCR primers 5'-AATTAAAGCG GCCGCCAGAT
TTGCTCTCAGATGCG-3' (forward) and 5'-TTAATTTGTC CTGAGGACTC AGGGC-3'
(reverse), thereby introducing a NotI restriction site into
the 5'-end of the product. After restriction digest with
NotI, the PCR fragment was labeled by a fill-in reaction
using Klenow polymerase (Roche Molecular Biochemicals) and
[ GST Pull-down Assays--
35S-labeled proteins were
obtained by in vitro translation of pcDNA3-based
expression vectors using T7 RNA polymerase and the TNT translation
system (Promega) according to the manufacturer's instructions.
GST-protein purification and GST pull-downs were performed as described
(46). The expression vectors for GST-Ets-1 fusion proteins have been described.
Flk-1 and HIF-2a Are Co-regulated during Development--
We
recently demonstrated that the endothelial bHLH PAS domain
transcription factor, HIF-2 Differential Activation of Flk-1 Promoter by HIF-2
To localize the domains within the HIF molecules that are responsible
for differential activation of the Flk-1 promoter, a series of HIF-2
We then tested whether HIF-1 HIF-2
We then performed DNase I footprint analysis of a 226 bp fragment from
the Flk-1 promoter ( HIF-2 HIF-2
We further addressed the question of what domain of the HIF-2 The EBS3/HBS1 Element of the Flk-1 Promoter Confers
Strong Inducibility by HIF-2
To test whether the EBS3/HBS1 element from the Flk-1 promoter is able
to confer endothelial cell-specific expression to heterologous promoters, a variety of cell lines was transfected with the
[EBS3/HBS1]2 tk-luc constructs and subjected to analysis
of reporter gene expression (Fig. 7B). The highest activity
of this construct was obtained in endothelial cells, followed by COS
and Q2bn cells. In CEFs, (chicken embryonic fibroblasts) 3T3
fibroblasts, and HepG2 cells the construct was less active, and it was
inactive in Hepa1-C4 cells, which lack functional ARNT/HIF-1 EBS/HBS Elements of the Flk-1 Promoter Are Required for
Endothelial Cell-specific Gene Expression in Vivo--
To assess
whether the EBS/HBS elements in the native Flk-1 promoter are necessary
for transcriptional activation in vivo, we sequentially
mutated the individual Ets and HIF-2 Signaling via the VEGF receptor, Flk-1, is thought to be limiting
for endothelial cell proliferation and survival and indispensable for
the initial development of the vascular system. Because expression of
Flk-1 is highly restricted to endothelial cells and tightly regulated
during development, characterization of the gene regulatory elements of
Flk-1 would provide insights into the mechanisms of both endothelial
cell-specific restriction and temporal regulation of gene expression.
In previous studies we identified a 430-bp minimal enhancer from
the first intron of the Flk-1 gene, which in conjunction
with the Flk-1 promoter is sufficient and necessary to drive
endothelial cell-specific gene expression in transgenic mice (19).
Although the enhancer partially conferred endothelial cell-specific
expression of the lacZ reporter gene to a heterologous promoter, full recapitulation of endothelial expression was obtained only in conjunction with the Flk-1 promoter (19). These data together
with the endothelial cell-specificity of the Flk-1 promoter (47)
indicate that the binding of endothelial cell-specific transcription
factors to the promoter is necessary to enhance the expression of
Flk-1.
In this study, we have identified two functional binding sites for
HIF-2 within the Flk-1 promoter, located in close proximity to
functional Ets consensus sites. These binding sites constitute positively acting elements that confer inducibility by HIF-2 Cooperative activation of the Flk-1 promoter is reflected by the
physical interaction between Ets-1 and HIF-2 Unlike its close relative HIF-2 It cannot be ruled out that HIF-2 It may be speculated that, depending on the genetic background HIF-1 In the present study we show that HIF-2 (HIF-2
) (but not its close
relative HIF-1
), cooperates with Ets-1 in activating transcription
of the vascular endothelial growth factor receptor-2 (VEGF-2) gene
(Flk-1). The receptor tyrosine kinase Flk-1 is
indispensable for angiogenesis, and its expression is closely regulated
during development. Consistent with the hypothesis that HIF-2
controls the expression of Flk-1 in vivo, we show here that
HIF-2
and Flk-1 are co-regulated in postnatal mouse brain
capillaries. A tandem HIF-2
/Ets binding site was identified
within the Flk-1 promoter that acted as a strong enhancer element.
Based on the analysis of transgenic mouse embryos, these motifs are
essential for endothelial cell-specific reporter gene expression. A
single HIF-2
/Ets element conferred strong cooperative induction by
HIF-2
and Ets-1 when fused to a heterologous promoter and was most
active in endothelial cells. The physical interaction of HIF-2
with Ets-1 was demonstrated and localized to the HIF-2
carboxyl
terminus and the autoinhibitory exon VII domain of Ets-1,
respectively. The deletion of the DNA binding and carboxyl-terminal
transactivation domains of HIF-2
, respectively, created dominant
negative mutants that suppressed transactivation by the wild type
protein and failed to synergize with Ets-1. These results
suggest that the interaction between HIF-2
and endothelial Ets
factors is required for the full transcriptional activation of Flk-1 in
endothelial cells and may therefore represent a future target for the
manipulation of angiogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(HIF-1
) and therefore was renamed HIF-2
(42). Like HIF-1
,
HIF-2
protein is stabilized by hypoxia and, as a heterodimer with
ARNT, binds to a six-base pair consensus sequence, the
hypoxia-responsive element (HRE) in the regulatory sequences of
hypoxia-responsive genes (40, 43). However, HIF-2
activates not only
the transcription of hypoxia-inducible genes such as VEGF and
erythropoietin (37, 40) but also of genes for endothelial
receptor tyrosine kinases, such as Flk-1 and
tie-2 (19, 40). Although in the erythropoietin enhancer and
the VEGF promoter HIF-2
apparently utilizes the HRE, Flk-1
expression was not activated by HIF-1
, and no classical HRE was
detected in the Flk-1 promoter.
collectively suggested that this transcription factor is a dual candidate regulator of vascular development by activating both components of the VEGF/Flk-1 signaling system. Therefore, we aimed to
analyze the mechanism by which HIF-2
activates transcription of
Flk-1. In this study, we found that HIF-2
(but not HIF-1
), although a relatively moderate activator of Flk-1 transcription, is
synergistic with Ets-1 in stimulating the Flk-1 promoter. HIF-2
and
Ets-1 (but not HIF-1
) physically interact via their carboxyl termini
and exon-VII domains, respectively. HIF-2 binds to two HRE-related
sequences, each in close proximity to functional Ets binding sites in
the Flk-1 promoter. These two pairs of transcription factor binding
sites constitute enhancer elements that confer strong inducibility by
HIF-2 and Ets-1 when fused to heterologous promoters. They are
indispensable, positively acting elements for the Flk-1 5'-flanking
region and are essential for endothelial cell-specific reporter gene
expression in transgenic mice. As HIF-2
protein and Flk-1 are found
to be co-regulated during development in brain capillaries, our data
emphasize the importance of a cooperative interaction of the HIF-2
and Ets transcription factors for vascular growth and differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cDNA was cloned in-frame into the SacI
site of the prokaryotic His tag expression vector, pQE31 (Qiagen). By
nickel affinity chromatography, an 87-kDa 6XHis-HIF-2
fusion protein
was purified from Escherichia coli cell lysates after
induction with isopropyl-1-thio-
-D-galactopyranoside
according to the instructions of the manufacturer (QIAexpressionist,
Qiagen). The protein was used for immunization of a rabbit according to standard protocols (Eurogentech), and immunoglobulin from sera was
affinity-purified over protein G-Sepharose (Amersham
Biosciences). The antiserum readily detected HIF-2
in the
nuclei and cell lysates of transfected cells and did not cross-react
with HIF-1
upon Western blot or immunohistochemistry of cells
transiently transfected with HIF-1
and HIF-2
, respectively. The
antiserum was applied at a dilution of 1:50-1:100 to cryostat sections
of postnatal mouse brain, and bound antibody was detected using the
Vectastain ABCTM Elite Kit (Vector Laboratories). Detection of Flk-1
protein was as described previously (44). For Western blot analysis, cell lysates from reporter gene assays were normalized for
Renilla luciferase units and separated on 8% sodium dodecyl
sulfate polyacrylamide gels. Proteins were transferred onto
nitrocellulose membranes (Schleicher & Schüll) using a semidry
blotting apparatus, and after blocking with bovine serum albumin, the
blot was incubated with the primary antibody. Bound antibody was
visualized with species-specific peroxidase-conjugated secondary
antibody (Dako) or protein G-peroxidase (Pierce) and ECLTM techniques
(Amersham Biosciences). Monoclonal anti HIF-1
and HIF-1
antibodies were from Novus Biologicals, and anti-Ets antibody (C275)
was from Santa Cruz Biotechnology. In situ hybridization was
performed on frozen sections using [35S]UTP-labeled
antisense RNA probes generated from a
PstI-HindIII fragment of HIF-2
cDNA as
described in detail previously (38).
-mercaptoethanol, and antibiotics. Hepa1-C4, COS-7, NIH3T3 fibroblasts, bovine aortic endothelial cells (BAE), and human umbilical
vein endothelial cells were cultured in DMEM-F12 supplemented with
fetal calf serum (Roche Molecular Biochemicals) and antibiotics. Endothelial cell media were supplemented with 0.4% (v/v) endothelial cell growth supplement (PromoCell). Chicken embryonic fibroblasts and
Q2bn cells were grown in the presence of 8% fetal calf serum (Roche
Molecular Biochemicals) and 2% chicken serum (Sigma). HepG2 cells were
grown in RPMI medium (Invitrogen). For induction of HIFs, cobalt
chloride (Sigma) to a final concentration of 100 µM was
added to HEK-293 and BAE cells 16 h before cells were harvested.
and HIF-2
cDNAs has been described previously (19).
cDNAs encoding full-length or mutated HIF-1
and HIF-2
proteins were generated using proofreading ExTaq DNA polymerase (Takara), and expression vectors were created by insertion of PCR products into the multicloning site of pcDNA3 (Invitrogen). By use of specific reverse PCR primers encoding the FLAG epitope followed by a stop codon and an appropriate restriction site, all
constructs were FLAG-tagged at their carboxyl termini. All mutations
were generated from the FLAG-tagged full-length clones. Deletion
mutants were prepared by targeting internal sequences with the
corresponding PCR primers. For deletion of the TAD N domain, an
internal forward primer was designed that bridged the TAD N-encoding
sequence and was used in combination with a vector-specific reverse
primer to amplify a cDNA fragment encoding the carboxyl terminus
with the mutation at its 5'-end. In a second PCR reaction, this
fragment was used together with vector-specific sequencing primers and
a 3'-truncated template to amplify the HIF-2
TAD N cDNA. The
HIF-2
/1
mutant was created using reverse complementary chimerical
primers matching the intended junction between the HIF-2
amino
terminus and the HIF-1
carboxyl terminus. These mutagenesis primers
were used in combination with vector-specific sequencing primers to
amplify HIF-2
5'- and HIF-1
3'-cDNAs, which in a second round
of PCR were used to create the fusion product. The mutants were cloned
into the KpnI and NotI sites of pcDNA3. All
expression plasmids were tested for functionality by in
vitro and in vivo translation and subsequent Western
blot analysis using FLAG tag-specific M2 antibody (Kodak, Integra
Biosciences) or protein-specific antibodies. The HIF constructs and
their corresponding amino acids were: HIF-1
, aa 1-822; HIF-2
, aa
1-874; HIF-2
dnb, aa 24(M/R)-874; HIF-2
dn, aa 24(M/R)-325;
HIF-2
TAD C, aa 1-783; HIF-2
TAD N, aa 485-538 deleted,
HIF-2
/1
, aa 1-318 of HIF-2
fused to aa 316-822 of HIF-1
.
The ARNT/HIF-1
cDNA (45) was cloned into the HindIII
and NotI sites of pcDNA3. The Ets-1 cDNA expression
vector was pSG5c-Ets1 (20). The expression plasmids of Ets-1 mutants
Ets238-441 and Ets331-441 have been described (46).
648 to +299 was ligated into the
KpnI-HindIII sites of pGL2 basic vector (Promega)
upstream of the promoter-less cDNA encoding firefly luciferase as
reporter. For testing of enhancer elements from the Flk-1 promoter,
synthetic double-stranded oligonucleotides including the EBS3 and HBS1
or HBS2 and EBS6 sites of the 5'-flanking region of the
Flk-1 gene were fused to the 145-bp minimal promoter from
herpes simplex thymidine kinase and inserted upstream of the firefly
luciferase gene into the SmaI site of pGL2 plasmid. Ets and
HIF-2
motifs were mutated by replacement by unrelated sequences. The
oligonucleotide sequences were: EBS3/HBS1, 5'-GCCCGGCACA GTTCCGGGGT AGTGGGGGAG TGGGCGTGGG AAACCGGGAA-3'; EBS3mut/HBS1, 5'-GCCCGGCACA GTCAATGGGT AGTGGGGGAG TGCGGCGTGG GAAACCGGGAA-3'; EBS3/HBS1mut, 5'-GCCCGGCACA GTTCCGGGGT AGTGGGGGAG TGCAGAGAGG
AAACCGGGAA-3'; and HBS2/EBS6, 5'-CCAGTGGGGG GCGTGGCCGG ACGCAGGGAG-3'.
Clones containing tandem repeats of the oligonucleotides were
verified by sequence analysis using GL-1 and -2 sequencing primers
(Promega) and were used for transient transfection in reporter gene assays.
and Ets core binding sites of interest
within the Flk-1 promoter luciferase construct. The HIF-2
core
binding sites, GGCGTG, and the Ets binding sites, CGGA, were
replaced by CAGAGA and ATTG, respectively. Mutations were confirmed by
DNA sequencing and constructs were used for transient transfection in
luciferase reporter assays. For generation of transgene constructs, the
wild type and mutated 946 bp KpnI-HindIII promoter fragments were inserted 5' of a promoter-less LacZ
cDNA, which was flanked at its 3'-end by the 2300-bp enhancer
element from the first intron of the Flk-1 gene (Fig.
8B). This construct was contained in a pGL2-based plasmid.
We have previously shown that the wild type promoter/enhancer construct
drives the expression of the lacZ reporter gene in
endothelial cells during embryogenesis (19, 20). The generation,
genotyping, and whole mount lacZ staining of transgenic mouse embryos
was performed as described (19).
80 °C until used.
-32P]ATP. Sequences are listed in Fig.
4B. Unlabeled oligonucleotides were added to the reaction
mixture in a ratio of 1:1-1:500 to the labeled probes. For supershift
analysis 25 µg of purified antibody or rabbit IgG was included into
the reaction. Protein-DNA complexes were separated on polyacrylamide
gels and visualized by autoradiography.
-32P]dCTP/dGTP. DNA sequencing and footprint
analysis of the labeled fragment were performed as described (48, 50)
using BAE nuclear extracts.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, is an activator of the VEGF
receptor-2/Flk-1 promoter (19). To assess a possible role of HIF-2
in the regulation of Flk-1 expression in vivo, we generated
a polyclonal antiserum against an HIF-2
-His fusion protein and
performed immunohistochemistry and in situ hybridization on
frozen sections of postnatal mouse brain from different stages,
comparing its pattern and time course of expression with that of the
VEGF receptor Flk-1 (Fig. 1). It was
shown previously that in rats and mice expression of Flk-1 is
down-regulated in postnatal life in brain capillaries and that this
down-regulation correlates with the decrease of the mitosis index of
endothelial cells (51). Although HIF-2
mRNA was expressed constitutively in postnatal brain capillaries, the protein was detected
in endothelial cell nuclei only until postnatal day 20 (P20). At
P8, when proliferation of brain endothelial cells is at its
maximum (52), a strong co-expression of HIF-2
and Flk-1 was observed
in brain capillaries, and HIF-2
was found translocated to the
nuclei. Later, HIF-2
protein expression was detected in astrocytes
and their end feet residing on the brain capillaries but was
largely reduced in capillaries and absent from nuclei. At P30, when
proliferation of brain endothelial cells is at its minimum (52),
expression of Flk-1 mRNA and protein is almost absent from
capillaries. Thus, the pattern and time course of HIF-2
expression
correlate with the growth of brain capillaries, suggesting that this
transcription factor regulates Flk-1 expression in the developing
brain.
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Fig. 1.
Expression of HIF-2
and Flk-1 in freshly fixed mouse brain sections from postnatal
day 8 (P8) (adolescent) and P30 (adult). HIF-2
mRNA
is constitutively expressed in brain capillaries as shown by in
situ hybridization with radiolabeled murine HIF-2
probe. In
contrast, HIF-2
protein is regulated during postnatal development in
parallel with Flk-1, as shown by immunohistochemistry with
anti-HIF-2
and anti-Flk-1 antibodies. Although HIF-2
protein is
detectable in endothelial cell nuclei at P8, labeling is absent from
nuclei at P30. In addition to a weak labeling of the
endothelial cytosol in the mature brain, HIF-2
protein is detectable
in processes of astrocytes. Magnification: 1000×.
and HIF-1
Is Due to Differences in the Carboxyl Termini--
In previous studies
it was shown that a 947-bp upstream sequence of the Flk-1
gene (referred to as Flk-1 promoter in the following) contains strong
positively acting elements (19, 47). Together with a 430-bp minimal
enhancer from the first intron, the Flk-1 promoter conveys complete
orthotope expression of the LacZ reporter in the endothelium of
transgenic mouse embryos (19). To analyze whether hypoxia-inducible
factors could be involved in Flk-1 expression, in a first series of
experiments we co-transfected HIF-1
and HIF-2
, respectively, with
a construct in which the firefly luciferase reporter gene is driven by
the Flk-1 promoter in HEK-293 cells in concentrations from 2 to 400 ng
of plasmid DNA/24-well (1.8 cm2 of subconfluent cell
monolayer). HIF-2
transactivated the Flk-1 reporter gene
construct more strongly than HIF-1
. The titration experiment
revealed that the transactivation effect was already saturated at 20 ng
of transfected DNA (Fig. 2A, upper
panel). In contrast, protein expression as estimated from the
Western blot analysis of cell lysates was not saturated (Fig. 2A,
lower panel). We then transfected the plasmids into different cell
lines to exclude the possibility that the observation could be cell line-specific. In all cell lines tested, the outcome was similar (Fig.
2B). The data indicate that Flk-1 activation by HIF-2
is executed via a cell lineage-independent mechanism. As expected, the
absolute basal activity of the Flk-1 promoter was low in most cell
lines tested. The strongest relative activation of the Flk-1 promoter
was obtained in HEK-293 cells. Therefore, this cell line was used for
all further experiments. To exclude the possibility that the difference
between HIFs was due to the larger instability of HIF-1
under
normoxic conditions, reporter assays were performed in the presence of
cobalt ions in the medium (which mimics induction of HIF-1
by
hypoxia) (67). Cobalt chloride at a
concentration of 50 µM enhanced the activation of the
Flk-1 promoter construct by HIF-1
and HIF-2
almost equally
(1.5-fold for HIF-2
and 2-fold for HIF-1
) (data not
shown). This enhancement also ruled out the possibility that the
plasmid-derived HIF-1
transcript was not translated into a
functional protein.
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Fig. 2.
Differential activation of Flk-1 reporter
gene by HIFs. A, HIF-2 and HIF-1
expression
plasmids were co-transfected with Flk-1-luciferase reporter plasmid in
HEK-293 cells, respectively. The amount of co-transfected expression
plasmid is indicated. The total amount of transfected plasmid DNA was
kept constant by adding empty vector up to 400 ng. All further
experiments were performed at saturation conditions by transfection of
200 ng of expression plasmid. In all reporter experiments of this study
pRL-TK vector containing the Renilla luciferase reporter
gene under the control of the HSV tk promoter was co-transfected as an
internal standard. Relative luciferase activity was plotted as the
ratio of reporter to standard. Data are the means from three
independent assays. Expression of HIF proteins was checked by Western
blotting as shown in the lower panel. Equal amounts of
protein, as assessed from Renilla luciferase units, were
loaded. B, HIF-2
and HIF-1
expression plasmids,
respectively, were co-transfected with Flk-1-luciferase reporter
plasmid in different cell lines. The luciferase read-out of vector only
transfected cells was arbitrarily set to 1.
mutants was created, including a chimera in which the entire carboxyl
terminus was exchanged between HIF-1
and HIF-2
(referred to as
HIF-2
/1
) (Fig. 3A). The
mutants were tested for their ability to transactivate the
Flk-1-luciferase reporter construct. Transactivation by the
HIF-2
/1
mutant was significantly lower than by HIF-2
wild
type, and in many assays it was at the level of HIF-1
or even
lower (Figs. 3B and 5). Thus, the specific transactivation
properties reside in a segment of the HIF-2
molecule
carboxyl-terminally to the PAS domains. Therefore, mutants were
created in which the amino- and carboxyl-terminal transactivation
domains (TAD N or TAD C, respectively) were deleted from the carboxyl
terminus of HIF-2
(referred to as HIF-2
TAD N and HIF-2
TAD C) (Fig. 3A). These domains have been characterized in HIF-1
(53) and are conserved in HIF-2
(54). Although the
HIF-2
TAD N mutant transactivated the reporter construct only to
a minor extent, the HIF-2
TAD C mutant was almost equally as
active as the wild type (Fig. 3B). Deletion of the
DNA-binding domain (HIF-2
dnb) abrogated the transcriptional
activation as well (Fig. 3B).
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Fig. 3.
Differential activation of Flk-1
reporter gene by HIFs depends on carboxyl-terminal domains.
A, schematic drawing of wild type HIF-2 , HIF-1
, and
the mutants used for the reporter gene assay. B,
HIF-2
expression plasmid was co-transfected with HIF expression
plasmids and Flk-1 luciferase reporter plasmid in HEK-293 cells as
indicated (filled bars). Open bars, relative
luciferase activity after transfection of the HIF-1
and HIF mutants
only. The promoter activity is expressed relative to that of stimulated
by HIF-2
in combination with the control vector (arbitrarily set to
100; hatched bars). Overexpression of ARNT/HIF-1
did not
overcome the dominant negative effects observed under co-transfection
with HIF-2
dnb and HIF-2
TAD N (not shown).
and the HIF-2
mutants were able to
compete with the wild type HIF-2
in a dominant negative manner. The
results of this competition assay are shown in Fig. 3B. Only
the HIF-2
dnb and HIF-2
TAD N constructs significantly reduced
activation of the Flk-1 promoter by wild type HIF-2
. These results
indicate that binding of HIF-2
to both DNA and the TAD N is required
for activation of the Flk-1 promoter. Because also HIF-1
reduced
transactivation, albeit weakly, and all mutants and HIF-1
were
readily translocated to the nucleus (as assessed by immunofluorescence;
data not shown), it was possible that the dominant negative effects
were due to a competition for the heterodimerization partner,
ARNT/HIF-1
, which is necessary for DNA binding. However, forced
over-expression of ARNT, which was substantiated by Western blot
analysis, was not sufficient to overcome the dominant negative effects
(data not shown).
Binds to HRE-related Sequences in the Flk-1 Promoter That
Are Located in Close Proximity to Functional Ets Sites--
To
identify potential binding sites for HIF-2
in the Flk-1 promoter, we
analyzed the DNA sequence using the MatInspectorTM algorithm (Transfac
data base, Gesellschaft für Biotechnologische Forschung,
Braunschweig, Germany). Two HRE-like GGCGTG motifs, which contained the
ARNT/HIF-1
-binding consensus sequence CGTG (42), were located at
140 and
78, respectively (in the following referred to as HBS1 and
HBS2, respectively). Both motifs were found in close vicinity to each
of two Ets binding sites, which were proven to be functional in
vitro in a recent study (20) (referred to as EBS3 and EBS6,
respectively). The location of EBS and HBS motifs in the Flk-1 promoter
is depicted in Fig. 4A. To
determine whether the putative HBS were able to bind the HIF-2 complex,
we prepared nuclear extracts from BAEs and performed electrophoretic
mobility shift assays using radiolabeled double-stranded oligonucleotides containing the putative binding sites and flanking sequences. Protein-DNA complexes were separated on polyacrylamide gels
and analyzed after autoradiography. The mutated control oligonucleotide as well as the HBS1 oligonucleotide and an oligonucleotide in which the
HBS site was replaced by a classical HRE produced a strong nonspecific
shift (Fig. 4B, indicated by "nsp" in the
upper part of the gel). This was competed by unlabeled
mutated as well as wild type oligonucleotide. The wild type and the HRE
oligonucleotide formed an additional, smaller complex. The
formation of this complex could be inhibited by an excess of unlabeled
wild type oligonucleotide only but not by an excess of mutated control
oligonucleotide (Fig. 4B). The intensity of the specific
shift was enhanced about 2-fold after the treatment of the BAE cells
with cobalt chloride before the nuclear extract was prepared (data not
shown). This indicated that a hypoxia-inducible factor was responsible
for the shifting. To examine whether HIF-2
from the BAE nuclear
extract forms the complex with the HBS1 oligonucleotide,
affinity-purified anti-HIF-2
antiserum was added to the reaction
mix, and an almost complete supershift of the complex resulted (Fig.
4C). Because the antibody did not cross-react with HIF-1
(not shown) and this supershift was not obtained with preimmune serum,
the data clearly indicate that HIF-2
from endothelial cell nuclei
can bind to the HBS1 motif of the Flk-1 promoter. Similar results were
obtained with an HBS2 oligonucleotide.
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Fig. 4.
Analysis of the Flk-1 5'-flanking region for
putative HIF-2 binding sites.
A, two potential HIF-2
binding sites, HBS1 and HBS2, with
the consensus binding motif GGCGTG were found by sequence analysis in
the Flk-1 promoter as indicated. The HBS are in proximity to the
previously detected functional Ets consensus sites, EBS3 and EBS6.
B, electrophoretic mobility shift assay for protein from BAE
nuclear extracts binding to the HBS1 consensus sequence. The coding
strand sequences for each of the double-stranded oligonucleotides are
the following: WT, wild type HBS; HRE, HBS
replaced by hypoxia-responsive element sequence; M, mutated
HBS. Unlabeled competitor was included in the reaction mixture in a
ratio of 1:1 to 1:500 to the labeled probe. nsp, nonspecific
shift; sp, specific shift; fpr, unbound probe.
C, supershift analysis of proteins forming a complex with
the HBS1 (WT) oligonucleotide. The specific shift
(sp) is supershifted (ss) after the addition of
anti-HIF-2
antibody (HIF2) to the reaction mixture but
not after the addition of preimmune serum (PIS).
D, DNase I footprint analysis of a Flk-1 promoter fragment
from bp
194 to +32 as referred to the transcriptional start site.
NE, nuclear extract from BAE (2 to 20 µg) was included in
the reaction mixture. Equal amounts of bovine serum albumin
(BSA) were added to control reactions. Open
arrowheads indicate DNase-hypersensitive sites. EBS and
HBS are same as indicated for panel A. S, GA sequencing
ladder (Maxam-Gilbert sequencing reaction).
194 to +32), which contains the HBS1 and -2 sites, using nuclear extracts from BAE cells. Both the HBS1 and the
HBS2 together with their adjacent EBS3 and -6, respectively, were found
to be protected by proteins from the nuclear extract from digestion by
DNase. Protection of the pairs of binding sites was accompanied by the
occurrence of new DNase I-hypersensitive sites flanking the footprints.
Details are indicated in Fig. 4D.
, but Not HIF-1a, and Ets-1 Cooperate to Activate the Flk-1
Promoter--
In view of the close proximity of their binding sites,
which are occupied by nuclear proteins in the footprint analysis, we next asked whether HIF-2
and Ets factors can cooperatively activate the Flk-1 promoter. Because in previous studies Ets-1 has been found to
be a strong activator of endothelial cell-specific promoters, the
co-transfection experiments were performed only with this family
member. The results of representative co-transfection reporter experiments in HEK-293 cells are shown in Fig.
5. The effect of co-transfection of
HIF-2
and Ets-1 was clearly more than additive (Figs. 5 and
8A). In contrast, HIF-1
did not influence transactivation by Ets-1 (Fig. 5). This failure could be attributed to the
carboxyl-terminal half of HIF-1
, because the HIF-2
/1
chimera,
which contains the carboxyl terminus of HIF-1
, also failed to
synergize with Ets-1 (Fig. 5). We addressed further the question of
whether the dominant negative mutants of HIF-2
could interfere
with activation by Ets-1. As shown in Fig. 5, none of the mutants
reduced transactivation of the Flk-1 reporter construct by Ets-1.
Interestingly, HIF-2
TAD C, like the wild type, acted
synergistically with Ets-1 to stimulate the Flk-1 promoter. In
contrast, HIF-2
TAD N did not co-activate. These data show that
DNA binding of HIF-2
and the integrity of TAD N but not of TAD C
domains are necessary for the interaction with Ets-1. The synergistic
effect and the co-localization of their binding sites were suggestive
of a physical interaction of HIF-2
and Ets-1 transcription factors
on the Flk-1 promoter.
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Fig. 5.
Interaction of HIF-2
with Ets-1 in activating Flk-1 luciferase
reporter gene. Co-transfection of Ets-1 expression plasmid (200 ng) and HIF expression plasmids as indicated (200 ng each; for
structure of the HIFs, see Fig. 3A) with Flk-1 luciferase
reporter plasmid. Open bars, relative luciferase values
after transfection of HIF constructs only (
) or after co-transfection
with Ets-1 plasmid (+). Promoter activity is expressed relative to that
after stimulation with Ets-1 in combination with control vector, which
was set to 100 (filled bars).
and Ets-1 Interact Physically in Vitro--
To assess
whether the transcription factors HIF-2
and Ets-1 can interact
physically, in vitro translated HIF-2
, HIF-1
, and two
HIF-2
mutants including the HIF-2
/1
chimera were subjected to
pull-down assays with a series of purified glutathione
S-transferase (GST)-Ets-1 fusion proteins. A schematic
drawing of the constructs used in this assay is shown in Fig.
6C. As shown in Fig.
6A, the HIF-2
wild type protein strongly interacted with
GST-Ets-1 fusion proteins that contained either the exon VII domain (aa
238-328) or the more amino-terminal portion (aa 124-236). The
DNA-binding domain (aa 333-441), as well as a long fusion protein
containing all three domains (aa 124
441) showed only very weak
interaction. In contrast, HIF-1
interacted only with the
amino-terminal portion of Ets-1 (aa 124-236) and, similar to HIF-2
,
very weakly with Ets-(124-236). The exon VII domain of Ets-1 is an
important autoinhibitory domain repressing Ets DNA binding by an
allosteric mechanism that can be relieved by interaction with other
partner molecules (55).
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Fig. 6.
HIF-2 and Ets-1
interact physically in vitro. A,
35S-labeled in vitro translated HIF-1
and
HIF-2
were assayed by GST pull-down for their ability to interact
with the domains of Ets-1 (p54ets) by exposure to the GST fusion
constructs of Ets-1 indicated in C. B,
interaction of Ets-1 exon VII GST fusion protein with radiolabeled HIFs
and HIF-2
mutants (as depicted schematically in C).
C, schematic drawing of the constructs used for the GST
pull-down interaction assays shown in A and B and
a summary table of the results.
protein is necessary for interaction with Ets-1 exon VII domain. As
shown in Fig. 6B, the ability of HIF-2
to interact with
the Ets-1 exon VII domain resides in the carboxyl-terminal part of the
protein, just downstream of the PAS domains, because a mutant deleted
from amino acid 325 can still weakly interact, whereas the substitution
of the entire HIF-2
carboxyl terminus (from aa 318) with the
corresponding region of HIF-1
abolished the interaction completely.
These data support the finding that HIF-2 but not HIF-1 cooperates with
Ets-1 to activate the Flk-1 promoter.
/Ets-1 to Heterologous
Promoters and Is Highly Active in Endothelial Cells--
The EBS/HBS
motifs in the Flk-1 promoter were identified as potential cis-acting
elements, which could be important for endothelial cell-specific
expression of Flk-1 during ontogeny. To assess the role of these
elements, double-stranded oligonucleotides representing Flk-1 promoter
sequences containing the EBS/HBS elements were synthesized and fused
upstream to the HSV tk promoter driving a luciferase reporter gene.
Clones containing tandem repeats of the elements were elected (referred
to as [EBS3/HBS1]2 tk-luc and [HBS2/EBS6]2
tk-luc) and used for luciferase reporter assays in HEK-293 cells. A
schematic drawing of these constructs is shown in Fig.
7A, left panel. In this way,
inducibility by HIF-2
and Ets-1 was conferred to a heterologous
promoter. As compared with the wild type tk-luc construct, which was
only slightly active and not inducible, relative induction by
HIF-2
/Ets-1 was about 100-fold when the EBS3/HBS1 element was
oriented as in the Flk-1 promoter and about 40-fold when oriented in
the opposite direction (Fig. 7A). Mutation of the
corresponding sites largely abolished inducibility by Ets-1 and
HIF-2
. Interestingly, cooperative activation was lost when the Ets
site was mutated. After mutation of the HBS1, however, cooperativity
was retained but inducibility by Ets-1 was also reduced (Fig.
7A). This observation suggests that binding to DNA may not
be absolutely necessary for HIF-2
to interact cooperatively with
Ets-1. Experiments with the [HBS2/EBS6]2 tk-luc constructs essentially revealed similar activation profiles, albeit to
a much lower extent (6-20-fold; Fig. 7A). Essentially the
same results were obtained when the SV40 minimal promoter was used instead of the HSV tk promoter. The SV40 promoter was less
suitable, because moderate activation of the empty construct itself by
Ets-1 was observed (not shown).
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Fig. 7.
Activation of EBS/HBS tk-luc fusion reporter
by HIF-2 and Ets-1. A, tandem
repeats of the EBS3/HBS1 and HBS2/EBS6 elements from the Flk-1
promoter (for sequence details, see "Experimental Procedures") were
fused to the minimal promoter of herpes simplex virus thymidine kinase
driving the firefly luciferase reporter gene. The constructs are
indicated on the left. HBS1 and EBS3 sites were mutated. The
constructs were co-transfected with HIF-2
and Ets-1 expression
plasmids in HEK-293 cells. The maximum value of reporter gene
activation was arbitrarily set to 100, and the other values were
expressed relative to this value. B, to test the
[EBS3/HBS1]2 tk-luc reporter construct for cell
line-specific inducibility, 200 ng of the plasmid was transfected into
the indicated cell lines, and luciferase activity was measured.
. The
latter result suggests that HIF-2
, which needs heterodimerization
with ARNT/HIF-1
for activity, is required for transactivation of the
[EBS3/HBS1]2 tk-luc construct. The data indicate that the
EBS3/HBS1 element is a positively acting element of the Flk-1 promoter,
which may contribute to restriction of gene expression to endothelial cells.
binding sites within the Flk-1
promoter-luciferase reporter construct and tested their inducibility by
Ets and HIF-2
in luciferase reporter assays before testing them in
transgenic embryos. Mutation of individual Ets or HIF-2
binding
sites resulted in a moderate but significant reduction of inducibility
(data not shown). Even the complete mutation of the EBS3/HBS1 element,
which was found to be a strong enhancer element, did not reduce the
inducibility of the promoter by more than 60%. This finding suggested
that the HBS2/EBS6 element also contributes considerably to the
transcriptional activation of the Flk-1 promoter. Consistently, after
mutation of both HBS/EBS motifs, the inducibility of the promoter by
Ets-1 and HIF-2
was almost completely abolished (Fig.
8A). We then introduced the
wild type and the mutated promoters into a lacZ reporter plasmid containing the enhancer element from the first intron
of the Flk-1 gene (Fig. 8B). We have previously
shown that this construct strongly recapitulates
endothelial cell-specific expression of the Flk-1 gene
during embryogenesis by lacZ reporter gene expression (19,
20). Mutation of the EBS3/HBS1 element did not alter the vascular
expression pattern of the reporter gene ([mut EBS3/HBS1], Fig. 8,
C and D, Table I).
Mutation of the HBS2/EBS6 element was needed in addition to abolish
expression of the reporter gene completely ([mut HBS1 + 2/EBS3 + 6];
Fig. 8, E and F, and Table I). In only
one double mutant embryo, faint endothelial and ectopic reporter gene
expression was seen. These data indicate that the Ets/HIF-2 binding
motifs are strong positively acting elements of the Flk-1 promoter,
which are necessary for endothelial cell-specific expression of Flk-1
in vivo. This promoter specificity, together with the
endothelial cell-specific expression of HIF-2
, which is
developmentally regulated concordantly with Flk-1 gene
expression, constitutes a novel candidate regulatory system of
angiogenesis.
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Fig. 8.
Reporter gene analysis of HIF-2 and
Ets-binding sites in the Flk-1 promoter in transient transfection
assays and transgenic mouse embryos. A, the EBS3/HBS1
and HBS2/EBS6 elements were mutated in the Flk-1 promoter luciferase
reporter plasmid, and inducibility of mutant promoters by HIF-2 and
Ets-1 was tested as compared with the wild type promoter
(wt) in transient co-transfection assays. mut
EBS3/HBS1, EBS3/HBS1 sites mutated;
mut HBS1 + 2/EBS3 + 6, EBS3/HBS1 and HBS2/EBS6
sites mutated. B, structure of the reporter construct used
for creating transgenic embryos. LacZ coding sequence is fused to the
promoter and the enhancer sequence from the first intron of the
Flk-1 gene (
646 to +299 bp and +3947 to +1677 to 3947 bp
of the Flk-1 gene, respectively) C and D, LacZ
staining of an E10.5 embryo expressing
-galactosidase under the
control of the wild type [mut EBS3/HBS1] Flk-1 promoter/enhancer
construct. D
F, E10.5 embryos transgenic for the [mut HBS1 + 2/EBS3 + 6] Flk-1 promoter/enhancer construct. Scale bar,
2 mm.
LacZ reporter gene expression in transgenic mouse embryos
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
Ets-1 and their cooperative effects to a heterologous promoter in an
insertion-independent manner. Inducibility of the Flk-1 promoter was
almost completely lost when these elements were mutated. Therefore,
these elements were likely to represent the sought positively acting
elements that are necessary for complete endothelial cell-specific
expression of Flk-1. Finally, by mutational analysis in transgenic
mouse embryos these elements were proved necessary for endothelial
cell-specific reporter gene expression in vivo. Interestingly, mutation of both the HBS2/EBS6 and EBS1/HBS2 elements was necessary to obtain the loss of endothelial cell-specific transgene
expression. These results suggest that the HBS/EBS elements may be used
alternatively in vivo, which is consistent with our finding
that both HBS readily bind HIF-2 from endothelial nuclear extracts and
are occupied by nuclear protein. The in vivo findings do not
explain the large difference in inducibility conferred by the HBS/EBS
elements when fused to heterologous promoters. It can be speculated
that the different distances between the neighboring HIF-2 and Ets
binding sites and/or the distance to the transcriptional start site are
critical parameters that influence the degree of inducibility conferred
by the enhancer elements.
. The physical interaction of Ets-1 and other Ets family members with a variety of
other transcription factors has been demonstrated and implicated in
differentiation processes mainly in hematopoiesis (36). Although Ets-1
interacts with the bHLH protein USF-1 at the DNA-binding domains (46),
our GST pull-down experiments indicate that the carboxyl-terminal
domain of HIF-2
strongly interacts with the exon VII domain of
Ets-1. This domain is known as a negative regulatory domain, which
needs to be conformationally changed to allow Ets-1 to bind DNA (55,
56). Thus, interaction of this domain with HIF-2
probably enhances
DNA binding of Ets-1 and thereby establishes the mechanism underlying
hyperadditive transactivation. The RUNT domain factor, AML1,
interacts with Ets-1 in a similar manner (55).
, HIF-1
is not able to interact
with the exon VII domain of Ets-1 functionally. By swapping the parts
of the HIF molecules, the failure of interaction could be attributed to
structural differences in the carboxyl-terminal halves. The fact that
deletion of the TAD N domain completely abrogated cooperativity
suggests that TAD N is necessary for the recruitment of Ets-1 to the
DNA via binding to the exon VII domain. In contrast, the TAD C domain
does not appear to be required for this interaction. All mutants
(HIF-2
dnb, HIF-2
dn, HIF-2
TAD N) that fail to interact
with Ets-1 are dominant negative over the wild type HIF-2
but do not
reduce the basal level of Flk-1 promoter stimulation by Ets-1.
Therefore, it can be concluded that endogenous HIF-2
is not
necessary for the basal activation of Flk-1 promoter by Ets-1. This is
further supported by the fact that high levels of basal activity were
obtained in HEK-293 cells, which lack detectable amounts of HIF-2
protein (43).
can also co-activate Ets-1 without
specific DNA binding, because deletion of the HBS1 from our tk enhancer
constructs did abrogate the induction by HIF-2
but not the
cooperativity with Ets-1 completely. In contrast, intact EBS3 is
absolutely required for induction by Ets-1 and synergism with HIF-2. If
this speculation holds true, HIF-2
would represent another example
of a DNA binding transcription factor that can transactivate via
cofactors without specific DNA binding (57). However, as shown by
deletion experiments, the DNA-binding domain of HIF-2
is
indispensable for synergism with Ets-1. The seemingly conflicting
observation, that a DNA-binding domain but not the specific recognition
sequence is needed for activity, may be resolved by the hypothesis that
HIF-2
requires binding to DNA for cooperative activation but, when
associated with Ets-1, not necessarily binding to its specific
recognition sequence.
can substitute for HIF-2
in endothelial cells in vivo. This could account for the varying outcomes of the HIF-2
knock-out in mice, such as the lack of any vascular phenotype and severe defects
in angiogenesis (58, 59). Whether the weak interaction of HIF-1
with
the amino terminus of Ets-1 (as observed in our GST-pull-down
experiments) is of functional relevance remains to be elaborated. In
addition, in endothelial nuclear extracts only little residual binding
activity to the Flk-1 HBS was detected after supershifting with
specific HIF-2
antiserum. This indicates that HIF-2
is the
predominant endothelial HIF.
protein is highly expressed
in brain capillary endothelial cells of mice during angiogenesis and is
localized to endothelial cell nuclei. The kinetics of endothelial cell
proliferation were best studied in the brain, and
down-regulation of Flk-1 was shown to precede the decrease in mitoses
of capillary endothelial cells in the brain of postnatal mice and rats
(51, 52). In parallel to Flk-1, expression of HIF-2
protein is
largely down-regulated in the cytosol and disappears from the nuclei of endothelial cells, although strong expression of mRNA is still retained. This finding indicates that HIF-2
is regulated
post-translationally in endothelial cells and raises the question of
the underlying mechanism. Induction of HIF-2
by hypoxia has been
proven and, similar to HIF-1
, is likely to depend on stabilization
of the protein under hypoxia. This is achieved via an
"oxygen-dependent degradation domain," which under
normoxia is targeted by hydroxylation of distinct proline residues to
the degradation via the ubiquitin-proteasome pathway (54, 60, 61).
Relative hypoxia in the growing brain may be an important factor that
contributes to stabilization of endothelial HIF-2
protein and
consecutively influences transcription of its target gene,
Flk-1. However, the data on a transcriptional induction of
Flk-1 by hypoxia are controversial (62-65) (51). In contrast to Flk-1,
the transcription of VEGF receptor-1/flt-1 is induced by
hypoxia via an hypoxia-responsive element within the promoter (64).
Whether flt-1, like Flk-1 and tie-2,
is another HIF-2 target gene, remains to be examined. Interestingly,
gene regulatory elements of both tie-2 and flt-1
contain functional Ets sites and, like many other endothelial genes,
these can be transcriptionally activated by Ets-1 (26, 27, 29).
The nature of the Ets factors that regulate expression of these genes
in vivo is still elusive. Ets-1, Ets-2, TEL, NERF2, and
other family members may be involved. The high functional redundancy of
Ets family members expressed in the endothelium could account for the
lack of a vascular phenotype after disruption of the Ets-1 gene (66). Whether HIF-2
can interact with other Ets family members
and whether cooperative interaction of HIF-2
with Ets factors is
also involved in the transcriptional regulation of other endothelial
genes, and therefore represents a general mechanism for specifying
endothelial gene expression, remain to be established.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Hugo Marti (Bad Nauheim,
Germany) for providing the ARNT/HIF-1 cDNA and the
staff of the Max-Planck-Institut für physiologische und klinische
Forschung transgenic facility (Bad Nauheim, Germany) for
generating transgenic mice.
![]() |
FOOTNOTES |
---|
* This study was supported by the Zentrum für Molekulare Medizin, Universität zu Köln, and the Max Planck Society and by grants Fl 223/2-1, Pl 158/4-1 and Br1336/2-2 from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** Present address: Neurologisches Institut, Deutschordenstr. 46, 60528 Frankfurt am Main, Germany.
To whom correspondence should be addressed: Bayer AG,
Herz-/Kreislauf-Forschung II, Aprather Weg 18a, D-42096 Wuppertal,
Germany. Tel.: 49-202-364732; Fax.: 49-202-364808; E-mail:
ingo.flamme.if@bayer-ag.de.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M211298200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor; ARNT, aryl hydrocarbon receptor nuclear translocator; HRE, hypoxia-responsive element; DMEM, Dulbecco's modified Eagle's medium; BAE, bovine aortic endothelial cell; TAD N and TAD C, amino- and carboxyl-terminal transactivation domains, respectively; aa, amino acid(s); PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; bHLH, basic helix-loop-helix; PAS, Period-ARNT-Single-minded; dnb, DNA-binding domain; dn, dominant negative; HBS, HIF-binding site; EBS, Ets-binding site; GST, glutathione S-transferase; HSV, herpes simplex virus; tk, thymidine kinase; luc, luciferase.
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REFERENCES |
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