From the Investigative Treatment Division, National
Cancer Center Research Institute East, Kashiwa, Chiba, 277-8577 Japan,
the § Institute of General Pathology and Oncology, Second
University of Naples, 80138 Naples, Italy, the ¶ Department of
Experimental Pharmacology, University of Naples, Federico II, 80131 Naples, Italy, and the
Department of Surgery, University of
Tokyo, 113-8654 Tokyo, Japan
Received for publication, September 13, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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Transcription of hypoxia-inducible genes is
regulated by hypoxia response elements (HREs) located in either the
promoter or enhancer regions. Analysis of these elements reveals the
presence of one or more binding sites for hypoxia-inducible factor 1 (HIF-1). Hypoxia-inducible genes include vascular endothelial growth
factor (VEGF), erythropoietin, and glycolytic enzyme genes.
Site-directed mutational analysis of the VEGF gene promoter revealed
that an HIF-1 binding site (HBS) and its downstream HIF-1 ancillary
sequence (HAS) within the HRE are required as cis-elements for the
transcriptional activation of VEGF by either hypoxia or nitric oxide
(NO). The core sequences of the HBS and the HAS were determined as
TACGTG and CAGGT, respectively. These elements form an imperfect
inverted repeat, and the spacing between these motifs is crucial for
activity of the promoter. Gel shift assays demonstrate that as yet
unknown protein complexes constitutively bind to the HAS regardless of the presence of these stimuli in several cell lines, in contrast with
hypoxia- or NO-induced activation of HIF-1 binding to the HBS. A common
structure of the HRE, which consists of the HBS and the HAS, is seen
among several hypoxia-inducible genes, suggesting the presence of a
novel mechanism mediated by the HAS for the regulation of these genes.
Most higher eucaryotes require oxygen to meet essential metabolic
demands including oxidative phosphorylation, in which oxygen serves as
the terminal electron acceptor in mitochondria. Low cellular oxygen
tension is seen in physiological conditions, such as high altitude and
physical exercise, and in pathological conditions including ischemia,
inflammation, and neoplasm. A variety of systemic and cellular
responses for homeostatic adaptations are provoked in these hypoxic
conditions, including erythropoiesis, vasodilatation, angiogenesis, and
glycolysis. Hypoxia activates the transcription of genes whose products
mediate these responses (1). Hypoxia-inducible genes within these
respective categories include erythropoietin (EPO)1 (2), vascular
endothelial growth factor (VEGF) (3, 4), inducible nitric-oxide
synthase (5), heme oxygenase 1 (6, 7), aldolase A (8, 9),
enolase 1 (9), glucose transporter 1, lactate dehydrogenase A (LDHA)
(8-11), and phosphoglycerate kinase 1 (8, 10).
The hypoxia response elements (HREs) of these genes have in common one
or more binding sites for hypoxia-inducible factor 1 (HIF-1). HIF-1 was
originally reported by Semenza and Wang (2) as a nuclear factor that
was induced by hypoxia and bound to the HRE in the EPO gene. HIF-1 is a
heterodimer composed of HIF-1 VEGF is a hypoxia-inducible gene whose regulation and function have
been studied extensively. VEGF plays a key role in physiological angiogenesis, as observed in tissue regeneration, and in
pathophysiological angiogenesis, as observed in wound healing, tumor
growth, metastasis, psoriasis, and diabetic retinopathy (13). VEGF
expression is regulated by a variety of stimuli including hypoxia,
cobaltous ion, nitric oxide (NO), growth factors, and cytokines (14). Although hypoxia is regarded as the most potent regulator of this gene,
NO has drawn a great deal of attention recently as a regulator of the
VEGF gene. However, the role of NO in VEGF expression remains inconclusive. There are some observations that NO down-regulates the
expression of VEGF in vascular smooth muscle cells and hepatoma cells
(15-17). In contrast, our previous studies report that NO induces VEGF
gene transcription in glioblastoma and hepatoma cells (18, 19).
Recently, Dulak et al. (20) demonstrated that endogenous NO
enhances VEGF synthesis in rat vascular smooth muscle cells.
Analysis of the VEGF promoter has uncovered that one HIF-1 binding site
(HBS) in its 5'-flanking region functions as a cis-element regulating the hypoxic induction of VEGF. Liu et al. (3)
suggest that not only this HBS, but also an adjacent sequence located immediately downstream within the HRE, is essential for the hypoxic activation of this promoter. We demonstrated that both elements are
indispensable to the transcriptional activation of the VEGF gene by NO
and hypoxia (19). However, no further structural or functional analyses
of this downstream sequence have been performed.
In this study, we demonstrate that this adjacent HIF-1 ancillary
sequence (HAS) is a novel cis-element for VEGF gene induction by NO and
hypoxia and that protein complexes constitutively bind to the HAS in
several cell lines. In addition, we show that a common structure of the
HRE, consisting of the HBS and the HAS, is widely seen among
hypoxia-inducible genes including VEGF and EPO genes and some
glycolytic enzyme genes.
Plasmids and Transient Transfection--
The sequence of pHRE
contains the 5'-flanking sequence of the human VEGF gene between
positions
Human glioblastoma A172, hepatoma Hep3B, cervical carcinoma Hela, and
green monkey kidney COS-1 cells were incubated in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.), supplemented with
10% fetal bovine serum, at 37 °C in humidified incubators. Five
µg of reporter plasmid and 1 µg of pSV-nlsLacZ were transfected into A172 cells in 10-cm dishes using 20 µl of Lipofectin (Life Technologies, Inc.) in serum-free Opti-MEM (Life Technologies, Inc.).
After incubation for 15 h, the medium was replaced with the
regular culture medium. The cells were harvested 36 h after medium
replacement and dissolved in 0.25 M Tris-Cl, pH 7.5. Prior to harvest, the cells were exposed either to normoxia (21%
O2) or hypoxia (1% O2) or to 0.5 mM
S-nitroso-N-acetyl-DL-penicillamine (SNAP) (dissolved in 0.1% Me2SO) or 0.1%
Me2SO for 12 h. Cell lysis was performed by four
freeze-thaw cycles. Luciferase and Electrophoretic Mobility Shift Assays (EMSAs)--
The sense
strands of oligonucleotides used in EMSA are as follows: wt HBS,
5'-CAGTGCATACGTGGGCTCCA-3'; wt HBS + HAS, 5'-
CAGTGCATACGTGGGCTCCAACAGGTCCTCTTCC-3'; wt HAS,
5'-GCTCCAACAGGTCCTCTTCC-3'; mutant HAS,
5'-GCTCCAAactcaCCTCTTCC-3'. Nuclear extracts were prepared as
described previously (21). In brief, A172, Hela, and COS-1 cell
extracts were harvested either 3 h after Me2SO (0.1%)
or SNAP (0.5 mM in 0.1% Me2SO) exposure or
8 h after normoxic (21% O2) or hypoxic (1%
O2) exposure. Hep3B cell extracts were harvested either
8 h after Me2SO or SNAP exposure or 12 h after
normoxic or hypoxic exposure. The collected cells were centrifuged at
270 × g for 10 min at 4 °C. The pellet was resuspended in buffer A (10mM Tris-HCl (pH 7.6), 1.5 mM MgCl2 10 mM KCl, 2 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl
fluoride, 1 mM Na3 VO4, and 2 µg/ml each of pepstatin A, leupeptin, and antipain) and kept
on ice for 10 min. The cell solution was then homogenized by pipetting
more than five times with a syringe, followed by centrifugation at
12,000 × g for 2 min at 4 °C. The pellet was
resuspended in ice-cold buffer C (0.42 M KCl, 20 mM Tris-HCl (pH 7.6), 20% glycerol, 1.5 mM
MgCl2, 2 mM dithiothreitol, 0.4 mM
phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 2 µg/ml each of pepstatin A,
leupeptin, and antipain) and rotated slowly at 4 °C for 30 min.
After centrifugation at 12,000 × g for 10 min, the
supernatant was stored at
Nuclear protein (5 µg) was incubated with 3 × 104
cpm of 32P-labeled double-stranded probes and 0.1 µg of
calf thymus DNA in modified buffer Z+ (25 mM
Tris-HCl (pH 7.6), 80 mM KCl, 0.2 mM EDTA, 20%
glycerol, 5 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 1.2 mM
Na3VO4, and 3 µg/ml each of pepstatin A,
leupeptin, and antipain) for 30 min at room temperature (22).
Electrophoresis was performed on 5% nondenaturing acrylamide gels at
25 mA at 4 °C. The gels were dried, and the radioactivity was
localized with a Bioimage Analyzer (Fuji Film, Tokyo, Japan). In
competition assays, excessive amounts of unlabeled competitors were
added 5 min prior to addition of the labeled probe.
Sequence Analysis with GenBankTM Data--
With the
aid of GenBankTM, we obtained the sequences of the HRE in
hypoxia-inducible genes and compared these to the core sequences of the
HBS and their ancillary sequences.
Statistical Analysis--
The results are shown as the
means ± S.E. The statistical significance was assumed at a value
of p < 0.05 by the use of the unpaired Student's
t test.
Mutational Analysis of the HIF-1 Binding Site and Its Ancillary
Sequence in the VEGF Gene--
The protein complexes containing a
heterodimer of HIF-1
Previously, we demonstrated that not only the HBS, but also its
downstream HAS, is essential for NO and hypoxic induction of the VEGF
reporter gene (19). To identify the extent of the HAS, located
downstream of the HBS, we tested the response of pHRE and its related
mutants (pHREm2a-pHREm2k) to NO and hypoxia. Fig.
2, A and B,
illustrates the results of the mutation analysis of the HAS. Because
pHREm2b, 2c, and 2d lost NO- and hypoxia-induced luciferase activity
(Fig. 2A), a 9-nt sequence, AACAGGTCC, was found to contain
the core of the HAS. Further analysis of the sequence requirement (Fig.
2B) revealed that a 2-base pair mutation within ACAGGT
(pHREm2g, 2h, and 2i) resulted in the loss of response, but a mutation
at the first A (pHREm2k) did not abrogate luciferase activity. A
mutation at TCC (pHREm2d), but not at CC (pHREm2j), within the sequence
ACAGGTCC attenuated the reporter activity. Thus, any substitution
within CAGGT eliminated the promoter response to either stimulus. This
result suggests that these 5 nt constitute the HAS, and the strict
sequence requirement might be indicative of the binding of some factor
to the HAS. These experiments indicate that NO and hypoxia similarly
enhance the VEGF promoter activity, and both the HBS and the HAS are
required as cis-elements for the activation of VEGF by these
stimuli.
Spatial Alignment of the HBS and the HAS--
The sequences ACGTG,
within the HBS, and CAGGT, the core sequence of the HAS, form an
imperfect inverted repeat. This raises a question whether a secondary
or tertiary structure formed by the above two elements is critical.
Therefore we prepared mutants containing either an inverted (pHREm3a)
or direct (pHREm3b) repeat of the HBS of the VEGF gene, whose spacer is
identical to that of the wild type (Fig.
3A). A pHRE mutant, with a
perfect inverted repeat of the HBS (pHREm3a), had a 2-fold greater
activity than that of the wild type (pHRE) (p < 0.01).
In contrast, a direct repeat of the HBS (pHREm3b) lost its
responsiveness to both stimuli (p < 0.01). The same
experiments were performed using mutants containing either an inverted
(pHREm3c) or direct (pHREm3d) repeat of the HAS of the VEGF gene. Both
mutants were found to be unresponsive to either stimulus. These results
suggest that a secondary structure of the HRE may be critical for the
promoter activity and that the HAS cannot compensate for the HBS.
The HBS and the HAS are located adjacent to each other. To investigate
whether these two elements function interdependently, we constructed
pHRE mutants, with either a 2-nt deletion within (pHREm3e), or a 5-nt
insertion (pHREm3f) into, the spacer and tested the reporter activity
after NO and hypoxic treatments. As shown in Fig. 3B, either
mutation resulted in a loss of reporter activation, suggesting that the
spacing between these motifs is crucial for the promoter activity. This
result raises the possibility that some putative factor, what we call
an HIF-1 ancillary factor (HAF), may bind to the HAS and interact with
HIF-1 for VEGF gene induction.
Characterization of Nuclear Proteins That Bind to the HAS of the
Human VEGF Promoter--
Hypoxia-induced HIF-1 activity mediates
transcriptional activation of the VEGF gene. HIF-1 forms DNA-binding
complexes containing the p300/ cAMP-response element-binding
protein when bound to its target HBS under hypoxic conditions
(25). However, no previous study has described binding factor(s) to the
adjacent HAS. To identify proteins that bind to the HAS of the VEGF
gene, we analyzed, in vitro, the binding of nuclear proteins
to three kinds of 32P-labeled oligonucleotides
corresponding to the HBS (wt HBS), the HAS (wt HAS), and both elements
(wt HBS + HAS) in NO- or hypoxia-treated cells. Nuclear proteins were
extracted from A172 cells cultured under 1% O2 for 8 h or 0.5 mM SNAP for 3 h or respective control cells. The extracts were incubated with labeled probes for 30 min at room temperature in the binding buffer Z+, and the mixtures were
electrophoresed on nondenaturing acrylamide gels. As shown in
Fig. 4A, EMSA revealed that
DNA-protein complexes (C1) were always present when either wt HBS or wt
HBS + HAS was used as a probe. These bands were observed with probes
reproducing the HBS of VEGF (3, 22) and represent constitutive binding
protein complexes. Doublet bands of less mobility (Fig. 4A,
H1 and H2) appeared only when nuclear extracts from the NO- or
hypoxia-treated cells were used. These bands include an HIF-1
heterodimer, which was assessed by supershift assays using antiserum
against either HIF-1
In competition assays, using wt HBS as a labeled probe, HIF-1 bands
induced by SNAP (Fig. 4B, H1 and H2) were displaced by excessive unlabeled wt HBS oligonucleotides but not by wt HAS. This
result suggests that these bands are specific for the HBS, but the HAS
is not a candidate for the binding site of HIF-1. The same result was
obtained with hypoxia-treated extracts (data not shown).
Fig. 4A also reveals the presence of constitutive bands (C2)
distinct from C1 when wt HBS + HAS or wt HAS was used as a probe. Another EMSA (Fig. 4C) shows that mutated HAS did not form
C2 protein bands, and these bands were displaced only by excessive unlabeled wt HAS oligonucleotides. These results demonstrate that protein complexes that represent C2 bands specifically bind to the HAS
of the human VEGF gene. Furthermore, this C2 band was not supershifted
by antibodies against HIF-1
Our mutational analysis of the VEGF promoter suggests a possible
interaction of HIF-1 and the HAS-binding factor for the VEGF gene
induction. To test if this HAS factor is present in cell lines where
HIF-1 is activated under hypoxic conditions, we performed EMSA by using
nuclear extract from A172, Hep3B, Hela, and COS-1 cells after normoxic
or hypoxic exposure. Fig. 4, A and D shows that
the HIF-1 band is induced only in NO or hypoxic nuclear extracts, whereas the HAS-binding complex is always visible and inhibited in part
by hypoxia and NO in all analyzed cell lines.
To analyze the influence of the HAS on HIF-1 binding activity to the
HBS, we performed another EMSA by using wt HBS as a labeled probe and
either wt HBS or wt HBS + HAS as competitors. As shown in Fig.
5, excessive unlabeled wt HBS and wt HBS + HAS similarly displaced NO-induced HIF-1 bands (H1 and H2) and
constitutive bands (C1), suggesting that the HAS does not significantly
influence the binding affinity of HIF-1 to the HBS. This is also the
case when using nuclear extracts from the hypoxia-treated cells (data not shown).
Structural Conservation of HREs in Hypoxia-inducible
Genes--
Hypoxia induces a number of genes whose promoter or
enhancer region contains one or more HBSs. They include aldolase A,
enolase 1, glucose transporter 1, LDHA, phosphofructokinase L,
inducible nitric-oxide synthase, phosphoglycerate kinase 1, heme
oxygenase 1, EPO, transferrin, and VEGF genes. Most of them have
(T/G)ACGTG as a consensus sequence for the HBS, although HBSs in
glucose transporter 1 (GGCGTG) and enolase 1 (TGCGTG) do not meet this consensus sequence perfectly (24).
We have determined the exact extents of the HBS and the HAS in VEGF and
found that both elements form an imperfect inverted repeat. Moreover,
the spacing of 8 nt in the VEGF gene is crucial. Surprisingly,
analysis of the HREs of the above genes revealed that the HREs in 7 of
11 hypoxia-inducible genes form an imperfect inverted repeat and that
the spacing is 8 nt in 6 genes and 9 nt in the rest (Fig.
6). These data suggest that a novel
common mechanism may exist, where a putative HAF has a pivotal role for induction of these genes.
Responses of the Human EPO Enhancer to NO and Hypoxia--
We have
demonstrated that the HAS is essential for VEGF gene induction and that
it is present in several hypoxia-inducible genes. To test whether NO
up-regulates the promoter function of other hypoxia-inducible genes and
whether the HAS functions as a cis-element in these genes, we prepared
reporter plasmids containing a wild-type HRE (pHREepo), a mutated HBS
(pHREepom1), and a mutated HAS (pHREepom2) of the EPO gene upstream of
the herpes simplex virus thymidine kinase promoter. These constructs
were transfected into A172 cells, and the luciferase activity of the
extracts was assayed. After treatments of either SNAP or hypoxia for
12 h, pHREepo was induced 10- and ~40-fold, respectively, when
compared with their respective controls. However, a mutation in either the HBS or the HAS almost completely abolished the response to either
stimulus (Fig. 7), indicating that NO
up-regulates EPO transcription and that the HAS functions as a
cis-element in the EPO gene.
Positive Cooperativity of the HBS and the HAS in Human VEGF Gene
Induction--
Previously, we demonstrated that NO and hypoxia
up-regulate transcription of the VEGF gene by enhancing HIF-1 binding
activity (19). This observation suggests that the mechanisms of
VEGF gene induction by these stimuli share common features and that HIF-1 has a central role in the transcriptional activation. Analysis of
the VEGF promoter reveals that deletion of the HRE completely abolishes
VEGF induction by NO and hypoxia. A further analysis of the HRE shows
that not only the HBS, but also its downstream HAS, is essential for
induction by these stimuli and that the AP-1 site is required for its
optimal response (19). Similar cooperativity among several domains
within the HRE was also reported in the EPO and LDHA genes.
In the case of the human EPO gene, the HBS, its adjacent sequence
CACAG, and the binding site for hepatic nuclear factor 4 are crucial
for the enhancer activity, and a mutation of either site abolished its
hypoxic response (2, 26). The promoter analysis of the human LDHA gene
revealed that a mutation in the HBS entirely abrogated the response to
hypoxia, and mutation in either its upstream ACGT or its downstream
cyclic AMP response element significantly, but not completely, reduced
the promoter activity (11). These data indicate that multiple factors
mediate transcriptional regulation of these genes through a complex
interaction among these factors.
An inverted repeat of half-sites spaced by some nucleotides is
recognized as a common structure of the response elements for nuclear
receptors. In principle, nuclear receptors dimerize in solution and
bind to their response elements as dimers, although they can interact
with and bind to the half-sites independently as monomers (27). The
dimerization often enhances the binding of receptors to their response
elements by stabilization of the receptor-DNA complex rather than by an
increase in the association rate (28). In these cases, a spacing
between the half-sites does not usually affect its function (28, 29).
Our EMSA result revealed that HIF-1 can bind to its response element in
the absence of the HAS. In addition, unlike nuclear receptors, positive
cooperativity in HIF-1 binding between the HBS and the HAS was not observed.
Protein Complexes That Specifically Bind to the HAS of the Human
VEGF Gene--
Our mutational analysis of the VEGF promoter revealed
that the core sequences of the HBS and the HAS form an imperfect
inverted repeat with a spacing of 8 nt. This observation suggested that HAF, a putative protein that binds to the HAS, might be identical or
similar to HIF-1. Although mutations that increase stability of an
inverted repeat enhance the reporter activity (Fig. 3A), changes in spacing between half-sites abolished the activity (Fig. 3B). Thus, the geometry of these motifs might be more
important than the secondary structure. A strict requirement of the HAS sequence and precise spacing between the two motifs indicate that a
putative HAF might interact with the HIF-1 heterodimer as a novel
transcriptional factor in NO- and hypoxia-induced VEGF expression.
We demonstrated specific protein binding to the HAS of the VEGF gene in
EMSA for the first time. These specific DNA-protein complexes are
present in either normoxic or hypoxic nuclear extracts from A172,
Hep3B, Hela, and COS-1 cells, but they are inhibited in part by hypoxia
and NO (Fig. 4, A and D). It may be that part of
the HAS-binding factors form protein complexes with HIF-1 under NO and
hypoxic conditions, whereas the rest constitute attenuated C2 bands.
The HAS-specific C2 complexes do not contain HIF-1 or CBP/p300,
both of which are known to be essential for VEGF gene induction. These
findings suggest that an unknown HAS-binding protein may function as a
basic transcriptional factor in a wide range of tissues and organs, in
contrast with tissue-specific regulation of hepatic nuclear factor 4 in
the Epo gene (26). Although we have no information concerning the
identity of the HAS-binding factor in the DNA-protein complex, its
characterization is now in progress.
The HAS-specific complex is more visible the lower the
concentration of potassium in the binding reaction in A172
cells.2 Even 250 times excess
of unlabeled wt HAS could not displace this band completely (Fig.
4C). Therefore, it is possible that the HAS-specific complex
may bind to its target DNA with relatively lower affinity, and the
amount of these proteins may be abundant, as compared with competitors.
Common Structures of HRE in Hypoxia-inducible Genes--
A
comparison of sequences of HREs (Fig. 6) shows that a similar
structure, where the HBS and the HAS form an imperfect repeat, is seen
among several hypoxia-inducible genes and that these motifs are spaced,
most commonly, by 8 nt. Given that CACGT instead of CAGGT in the HAS of the VEGF gene functions as a
cis-element for the promoter activity, all the HASs can be described as
either CACG(T/C) or CACA(G/T) sequences (Fig. 6). In mutational
analysis of the HAS of VEGF, pHREm3a, which contains an inverted repeat of the wild-type HBS (CAGGTC'CACGTA), had a
2-fold greater activity as compared with pHRE (Fig. 3A), and
a pHRE-related mutant containing the HAS of LDHA or heme oxygenase 1 (CAGGT'CACGT) had an identical response to pHREm3a. In
contrast, an exchange of CAGGT (the HAS of VEGF) with CACAG
(the HAS of EPO) in the VEGF promoter abrogated induction by NO and
hypoxia.2 These results indicate that the effect on
increasing inducibility of the reporter gene by hypoxia and NO is
mediated by a gene-specific inverted HBS rather than by a consensus HAS
sequence. The C2 complex, as seen in Fig. 4, could not be detected in
EMSA by using probes that contained the HAS of LDHA and
EPO.2 These findings suggest that HAS-binding
factors of VEGF may be distinct from those of LDHA and EPO.
All of these HASs share a common sequence, CAC, except for the VEGF
gene. CAC in the sense or GTG in the antisense strand is a recognition
site of ARNT, as seen in the HBS ((T/G)ACGTG). ARNT is known as the
central dimerization partner for basic
helix-loop-helix-per-arnt-sim family transcription
factors including HIF-1 Conclusions and Implications--
In summary, a detailed analysis
of the VEGF gene promoter reveals that the sequences TACGTG and CAGGT
are the cores of the HBS and the HAS, respectively, and that both sites
are essential for the up-regulation of VEGF expression by NO and
hypoxia. We also show that the constitutive protein complex binds to
the HAS of VEGF. A similar structure of the HRE is seen among several hypoxia-inducible genes, indicating that a common mechanism may exist
in which an HAF-mediated pathway positively regulates the transcription
of these genes. We could not identify HAF in this study, but it may
interact with, or rather collaborate with, HIF-1 for the promoter
activation. VEGF plays a central role in tumor growth, progression, and
metastasis by enhancing its angiogenesis and vascular permeability. If
the HAF of VEGF is unique and distinct from that of other genes,
inhibition of HAF function would be able to suppress VEGF
induction without affecting expression of other hypoxia-inducible genes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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and HIF-1
(aryl hydrocarbon nuclear
translocator, ARNT) subunits, both of which belong to the basic
helix-loop-helix-per-arnt-sim family, and its
activity is tightly regulated by cellular oxygen tension (12).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
1014 and
903 relative to the transcription start site.
This segment, which contains the HRE, was prepared by polymerase chain
reaction amplification with primers 5'-CGTGGATCCAGCTGCCTCCCCCTTTG-3'
(sense strand) and 5'-GCCTCGAGGAGAACGGGAAGCTGTGTGG-3' (antisense
strand) and inserted between BamHI and XhoI of
pT81luc0 (19). The reporter pT81luc0 contains the herpes simplex virus thymidine kinase promoter, upstream of the luciferase-coding sequence. A series of pHRE-related mutants were prepared by cloning polymerase chain reaction-amplified segments, which contained substituted, deleted, or inserted sequences within the HRE, into the same site of
pT81luc0. A SV40-driven plasmid pSV-nlsLacZ, containing the
-galactosidase gene, was utilized for normalization of luciferase activity of the reporter plasmids (19). A reporter plasmid pHREepo and
its related mutants were prepared by cloning a segment of the
3'-flanking sequence of the human EPO gene (positions 3061 and 3116),
including either the wild-type HRE or a mutated HBS (TACGTG
TACtgt)
or a mutated HAS (ACACAG
AacaAG), into the same site of pT81luc0 as pHRE.
-galactosidase activities were
assayed as described before (19). The relative luciferase activity was
calculated as luciferase activity divided by
-galactosidase
activity. Fold induction was expressed as ratios of relative luciferase
activity of either SNAP/Me2SO or hypoxia/normoxia cells.
Protein concentration was determined by Bio-Rad protein assays
(Bio-Rad).
70 °C for EMSA.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HIF-1
were found to bind to the HBS in
the VEGF promoter. The HBSs are located in the promoter or enhancer
regions of several hypoxia-inducible genes, and the consensus sequence
was described as either 5'-(G/C/T)ACGTGC(G/C)-3' (3) or 5'-RCGTG-3'
(23). To determine the exact extent of the HBS in the human VEGF gene, a series of pHRE mutants with 1-3 nucleotide (nt) exchanges
(pHREm1a-pHREm1k) were synthesized, and their reporter expression was
compared with that of the wild type (pHRE) after NO and hypoxic
treatments. The pHRE mutants pHREm1b, 1c, 1e, 1f, and 1g, which
encompass the substitution of a 6-nt sequence, lost their response to
both 0.5 mM SNAP and hypoxia (1% O2) (Fig.
1, A and B).
Therefore, the 6-nt sequence, TACGTG, contains the core of the HBS. In
addition, a mutation at the last G (pHREm1i) of this sequence
eliminated its response to both stimuli, whereas a mutation at T, the
first nucleotide of TACGTG (pHREm1h), partially attenuated the activity of the luciferase reporter (Fig. 1B). Thus, at least, the
sequence ACGTG is required for the HBS to function. To clarify the
importance of the initial T within the sequence, TACGTG, we substituted
the T with an A, C, or G and tested the reporter activity of the
resultant constructs. Fig. 1C shows that the promoter
responded better to NO and hypoxia if the first nucleotide was a T or G
rather than an A or C (p < 0.05). A compilation of
HBSs (24) showed that the HRE contains a sequence (T/G)ACGTG as
a functional HBS in many hypoxia-inducible genes, whereas A or C in the
first nucleotide of this sequence is found in only a few genes. This
finding is consistent with our result that the sequence (A/C)ACGTG was
less functional (Fig. 1C). Together, our findings suggest
that a 6-nt sequence, TACGTG, is the core motif of the HBS in the VEGF
promoter.
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Fig. 1.
The HIF-1 binding site of the VEGF gene is
determined as TACGTG. A172 cells transiently transfected with
reporter plasmids and pSV-nlsLacZ were harvested after a 12-h
exposure to normoxia (21% O2), hypoxia (1%
O2), Me2SO (0.1%), or SNAP (0.5 mM
in 0.1% Me2SO). Results were expressed as the mean ± S.E. of six independent experiments. Nucleotides of putative HBS and
HAS are underlined, and substituted bases are shown in
lowercase letters. A, a series of pHRE-related
mutants with 3-nt substitutions within the HRE were prepared, and their
responses to SNAP and hypoxia were tested. *, p < 0.01 versus pHREm1a and pHREm1d. B, a series of
pHRE-related mutants with 1- or 2-nt substitutions within the HRE were
prepared, and their responses to both stimuli were tested. *,
p < 0.01; **, p < 0.05 versus pHRE. C, a series of pHRE-related mutants
with substitution of the first nucleotide of TACGTG were prepared, and
their responses to both stimuli were tested. *, p < 0.01; **, p < 0.05 versus pHRE; #,
p < 0.05 versus pHREm1h and pHREm1j.
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Fig. 2.
The HIF-1 ancillary sequence of the VEGF gene
is determined as CAGGT. A172 cells transiently transfected with
reporter plasmids and pSV-nlsLacZ were harvested under the same
conditions as in Fig. 1. Results were expressed as the mean ± S.E. of six independent experiments. *, p < 0.01; **,
p < 0.05 versus pHRE. Nucleotides of
putative HBS and HAS are underlined, and substituted bases
are shown in lowercase letters. A, a series of
pHRE-related mutants with 3-nt substitutions within the HRE were
prepared, and their responses to SNAP and hypoxia were tested.
B, a series of pHRE-related mutants with 1- or 2-nt
substitutions within the HRE were prepared, and their responses to both
stimuli were tested.
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Fig. 3.
The spatial alignment of the HBS and the HAS
is crucial for NO and hypoxic induction of the VEGF gene. A172
cells transiently transfected with reporter plasmids and
pSV-nlsLacZ were harvested under the same conditions as in Fig.
1. Results were expressed as the mean ± S.E. of six independent
experiments. *, p < 0.01 versus pHRE.
Substituted or inserted bases are shown in lowercase
letters. A, pHRE-related mutants with an inverted or
direct repeat of the HBS or the HAS were prepared, and their responses
to SNAP and hypoxia were tested. The closed and open
arrows indicate the HBS and the HAS, respectively. The
arrowheads arbitrarily indicate the orientation of the
half-site. B, pHRE-related mutants with either a 2-nt
deletion or a 5-nt insertion within the spacer between the HBS and the
HAS were prepared, and their responses to both stimuli were tested.
Nucleotides of putative HBS and HAS are underlined.
or HIF1
(ARNT) (19). Similar patterns of H1
and H2 bands were observed when nuclear extracts from the NO- or
hypoxia-treated cells were used with wt HBS and wt HBS + HAS
probes.
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Fig. 4.
The HAS is not a binding site for HIF-1, but
specific protein complexes bind to the HAS of VEGF in several cell
lines. Oligonucleotides for EMSA were as follows: wt HBS, which
contains the wild-type HBS, but not the HAS; wt HBS + HAS, which
contains the wild-type HBS and HAS; and wt HAS, which contains the
wild-type HAS but not the HBS. Cells were exposed either to 0.1%
Me2SO or 0.5 mM SNAP (in 0.1%
Me2SO) or to normoxia (21% O2) or hypoxia (1%
O2). Nuclear extracts (5 µg per lane) from these cells
were incubated with 32P-labeled probes in the presence or
absence of competitors prior to electrophoresis. DNA-protein complexes
are indicated by arrows. Hypoxia- or NO-induced complexes
with slower (H1) and faster (H2) mobility,
constitutively binding complexes to the HBS (C1) or HAS
(C2), and nonspecific bands (NS) are indicated.
( ), no serum; N, normoxia; H,
hypoxia; D, Me2SO; S, SNAP.
A, comparison of DNA binding activities recognized by wt
HBS, wt HBS + HAS, and wt HAS in A172 cells. B, competition
assays with either Me2SO- or SNAP-treated nuclear extracts
from A172 cells using wt HBS as a labeled probe in the presence of a
0-, 50-, or 250-fold molar excess of competitors. C, binding
specificity of the C2 band to its target HAS in A172 cells. Competition
assays were performed in the presence of a 250-fold molar excess of
unlabeled oligonucleotides. mut, mutant.
D, comparison of DNA binding activities recognized by wt HBS + HAS in normoxic (N) or hypoxic (H) extracts
from A172, Hep3B, Hela, and COS-1 cells.
, HIF-1
, or CBP/p300 (data not
shown), suggesting that the C2 band is irrelevant to these
transcriptional factors.
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Fig. 5.
The HAS has no effect on HIF-1 binding
affinity to the HBS. Competition assays, with SNAP-treated nuclear
extracts using wt HBS as a labeled probe, were performed in the
presence of a 0- to 28-fold molar excess of competitors.
Nitric oxide-induced complexes with slower (H1) and faster
(H2) mobility and constitutively binding complexes to the
HBS (C1) are indicated.
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Fig. 6.
The HREs of several hypoxia-inducible genes
contain a common structure consisting of the HBS and the HAS. The
sequences that contain the HBS and the HAS of several hypoxia-inducible
genes are shown. Note that these two motifs are usually spaced by 8 nt,
and all HASs, except for VEGF, contain CACG(T/C) or CACA(G/T). The
closed and open arrows indicate the HBS and the
HAS, respectively. The arrowheads arbitrarily indicate the
orientation of the half-site. Underlined nucleotides match
the corresponding nucleotides in the remaining half-site as an inverted
repeat.
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Fig. 7.
Nitric oxide up-regulates the EPO reporter
activity via the HBS and the HAS. A172 cells transiently
transfected with reporter plasmids and pSV-nlsLacZ were
harvested under the same conditions as in Fig. 1. The reporters
pHREepo, pHREepom1, and pHREepom2 contain the wild-type HRE, a mutated
HBS, and a mutated HAS of the human EPO gene, respectively. Results
were expressed as the mean ± S.E. of six independent experiments.
*, p < 0.01 versus pHREepo. Nucleotides of
putative HBS and HAS are underlined, and substituted bases
are shown in lowercase letters.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, endothelial per-arnt-sim homology domain protein 1, arylhydrocarbon receptor, and Sim
(30). It is possible that ARNT dimerizes with a member of the
per-arnt-sim homology family and binds to its target
HAS. Taken together, these results suggest that a common mechanism,
other than an HIF-1-mediated pathway, may exist for NO- and
hypoxia-induced expression of the hypoxia-inducible genes.
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FOOTNOTES |
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* 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.
** To whom correspondence should be addressed: Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, 277-8577 Chiba, Japan. Tel.: 81-471-34-6857 or -6880; Fax: 81-471-34-6866; E-mail: hesumi@east.ncc.go.jp.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008398200
2 H. Kimura, A. Weisz, T. Ogura, Y. Hitomi, Y. Kurashima, K. Hashimoto, F. D'Acquisto, M. Makuuchi, and H. Esumi, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: EPO, erythropoietin; VEGF, vascular endothelial growth factor; LDHA, lactate dehydrogenase A; HRE, hypoxia response element; HIF-1, hypoxia-inducible factor 1; ARNT, aryl hydrocarbon nuclear translocator; HBS, HIF-1 binding site; HAS, HIF-1 ancillary sequence; SNAP, S-nitroso-N-acetyl-DL-penicillamine; EMSA, electrophoretic mobility shift assay; wt, wild-type; nt, nucleotide(s); HAF, HIF-1 ancillary factor.
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