From the
Vascular endothelial growth factor (VEGF), a potent angiogenic
factor and endothelial cell-specific mitogen, is up-regulated by
hypoxia. However, the mechanism(s) responsible for hypoxic induction of
VEGF has not been clearly delineated. We report that the steady state
VEGF mRNA levels are increased 12 ± 0.6-fold, but the
transcriptional rate for VEGF is increased only 3.1 ± 0.6-fold
by hypoxia in PC12 cells. In order to investigate
cis-regulatory sequences which mediate this response to
hypoxia, we cloned the rat genomic sequences encoding VEGF and
identified a 28-base pair element in the 5` promoter that mediates
hypoxia-inducible transcription in transient expression assays. This
element has sequence and protein binding similarities to the
hypoxia-inducible factor 1 binding site within the erythropoietin 3`
enhancer. Post-transcriptional mechanisms have also been suggested to
play a role in the hypoxic induction of VEGF. Evidence is provided that
a frequently used polyadenylation site is 1.9 kilobases downstream from
the translation termination codon for rat VEGF. This site is 1.5
kilobases further downstream from the polyadenylation site previously
reported for VEGF. This new finding reveals sequence motifs in the
3`-untranslated region that may mediate VEGF mRNA stability.
The maintenance of an adequate supply of oxygen to the body
tissues is vital to survival. Increased respiratory rate
(1) and
increased red blood cell mass
(2) have been recognized as
important adaptations to a reduced environmental oxygen tension.
Similarly, at the level of a given tissue or organ, new blood vessel
formation has been recognized as an adaptive response to cellular
hypoxia
(3) . Hypoxia has been shown to be a very important
stimulus for the new vessel formation seen in coronary artery
disease
(4) , tumor angiogenesis
(5, 6) , and
diabetic neovascularization
(7) . VEGF,
The VEGF gene is
induced by hypoxia with characteristics that resemble the hypoxic
induction of the erythropoietin (Epo) gene
(11, 12) .
Expression of both genes is induced by cobalt and manganese, but not by
cyanide or azide, and hypoxic induction is inhibited by the protein
synthesis inhibitor cycloheximide
(6) The implication of these
studies is that there may be fundamental similarities in the oxygen
sensing pathway leading to the activation of these two genes. The
hypoxic induction of the Epo gene appears to be regulated by both
transcriptional and post-transcriptional
mechanisms
(16, 17, 18) . Hypoxia-inducible
factor 1 (HIF-1) has been identified as one of the proteins that
specifically binds to an enhancer element 3` to the Epo gene in a
hypoxia-regulated fashion
(19, 20) . Functional HIF-1
sites have recently also been reported in a number of hypoxia-regulated
genes, namely the genes for the enzymes phosphoglycerate kinase 1,
lactate dehydrogenase A, aldolase A, phosphofructokinase L, enolase 1,
and pyruvate kinase M
(21, 22) .
We have sought to
elucidate the mechanism of the hypoxic induction of VEGF steady state
mRNA by several methods. First, nuclear runoff experiments were
performed to measure the transcription rate of VEGF under normoxic and
hypoxic conditions. Second, in order to identify
cis-regulatory elements that mediate transcriptional
activation of VEGF by hypoxia, rat genomic sequences for VEGF were
cloned, and the promoter was analyzed using transient transfection
reporter assays. The sequence and protein binding characteristics of
one such regulatory element are described and compared to the hypoxia
regulatory element within the Epo 3` enhancer. Finally, the apparent
discrepancy between the size of VEGF mRNA by Northern blot analysis and
the published transcription termination site
(8, 23) for
VEGF mRNA is clarified. The role of sequence motifs in the
3`-untranslated region that are revealed by these studies are discussed
with regard to their ability to mediate changes in mRNA stability.
A control plasmid was not co-transfected
with the test plasmid. We have not found any significant difference in
luciferase activity between PC12 cells grown under 1% or 21% O
Fig. 5A illustrates the fold of hypoxic induction of
luciferase activity obtained from parallel tissue culture plates
transfected with the different pxp2 constructs and cultured at 1% or
21% O
In the present study we have demonstrated that hypoxia
increases the transcription rate of the VEGF gene. We have identified
one cis-regulatory element which partially mediates this
effect. However, the 3-fold increase in transcription rate does not
appear to be sufficient to account for the 12-fold increase in steady
state mRNA levels induced by hypoxia. Evidence for post-transcriptional
regulation by hypoxia has also been described for the Epo
(17, 18) and tyrosine hydroxylase genes
(41) . We have
identified sequences in the 3`-UTR of rat VEGF which may mediate
changes in mRNA stability.
Deletion and mutation analysis of the rat
VEGF 5`-flanking region revealed a functional HIF-1 binding site.
However, it is not possible to conclude from these studies that the
28-bp sequence containing the HIF-1 binding site accounts for all of
the transcriptional activation of the VEGF gene by hypoxia since the
induction seen with W28 5` to the minimal promoter is significantly
less than that observed with 1.7 kb of promoter sequence.
Interestingly, a 5-bp element in the Epo enhancer (5`CACAG3`) that is
essential for hypoxic induction of Epo
(20) is conserved in the
same orientation and position to the VEGF HIF-1 binding site identified
in these studies. In addition to band-shifting a hypoxia-inducible
species, the VEGF HIF-1 site binds a constitutive factor analogous to
the Epo enhancer. The significance of the finding that AP-1
oligonucleotide can compete for this constitutive factor will require
further studies but is intriguing in light of the close proximity of
the HIF-1 site and AP-1 site in the VEGF promoter. One testable
hypothesis is that this constitutive factor is AP-1.
It is likely,
however, that other cis-elements and trans-acting
factors are involved in the hypoxic induction of VEGF, as well. One of
these factors may be SP1. The VEGF minimal promoter, which contains 3
SP1 sites, is hypoxia-inducible in the transient transfection reporter
assay. Wu et al.(42) has demonstrated that binding of
the SP1 transcription factor is dependent on its redox state, with a
reducing environment (which occurs with hypoxia) stimulating SP1
binding as detected by band shift in an EMSA assay and functionally in
transient expression reporter assays. Similarly, the transcription
factor AP-1 has been shown to be a redox-sensitive transcription
factor
(43) .
Minchenko et al.(32) have
described the presence of hypoxic regulatory elements 5` and 3` to the
human VEGF gene. We cannot rule out that some of the hypoxia
responsiveness that we have been unable to attribute to the HIF-1 site
in the rat VEGF 5` promoter is due to the 5` element described by
Minchenko et al.(32) . However, alignment of rat and
human sequences from the region described by Minchenko et al.(32) demonstrate that this area is poorly conserved in the VEGF
flanking sequences. Our data are also in disagreement with those
reported by Minchenko et al.(32) regarding the 3`
element. We have demonstrated that the 3` sequences used by Minchenko
et al.(32) are in fact exonic rather than flanking the
VEGF gene as originally described. We have been unable to demonstrate
that this region, when placed 5` to the minimal promoter or 5` to the
thymidine kinase promoter, confers hypoxia responsiveness in transient
transfection assays. In both cases, this discrepancy might be explained
by species differences and the use of different cell lines and vectors
for the transient transfection assays.
Preliminary experiments with
the RNA polymerase II inhibitor actinomycin D
(44)
The
VEGF mRNA has been reported to be 3.7 kb by numerous investigators (8,
11, 23, 35, 45, 46) in a variety of cell and tissue types. The 3`-UTR
of the VEGF mRNA has previously been reported to be approximately 400
bp in length based on the finding of a poly(A) tract at the 3` end of a
cDNA clone and the identification of a nearby consensus polyadenylation
signal
(8, 23) . Transcription termination at this site
would yield a mRNA of only 2.2 kb. Indeed, a species of this size is
seen after long exposures of Northern blots probed with VEGF cDNA.
However, we have demonstrated by Northern blot analysis that
approximately 1.5 kb of sequence 3` to this first polyadenylation
sequence is frequently exonic. In addition, a potential polyadenylation
signal (A4) is located in the region mapped as the transcription
termination site. Use of this newly identified polyadenylation signal
would yield a VEGF mRNA transcript of
The sequences within this
3`-UTR include a number of sequence motifs that have previously been
demonstrated to be involved in the regulation of mRNA stability. AUUUA
sequences have been shown to mediate changes in mRNA stability for
numerous cytokines
(39, 40) , most notably interleukin-1
(47) and granulocyte macrophage colony stimulating factor (39,
48). Interestingly, the AUUUA sequences may mediate not only decreases
in mRNA stability but increases as well
(47) . It has been
postulated that a post-translational modification of the trans-acting
factors that bind to these sequences, such as a phosphorylation event
or redox shift, may explain this duality of function
(49) .
Alternatively, different AUUUA binding proteins may carry out different
functions. Nine canonical AUUUA sequences, one AUUUUA sequence, and one
AUUUUUA sequence are present in the 3`-UTR of VEGF as described here.
The role of these sequences in regulating the stability and the change
in stability of the mRNA by hypoxia is presently under investigation.
Polypyrimidine tracts, defined as pyrimidine-rich sequences, have
also been implicated in the increased mRNA stability of tyrosine
hydroxylase mRNA upon exposure to hypoxia
(41) . Four such tracts
of greater than 85% pyrimidine nucleotides and at least 28 bp in length
are found in the 3`-UTR of the VEGF gene. The role of these sequences
in regulating the increase in VEGF mRNA stability is also being
addressed by ongoing studies.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
(
)
also known as vascular permeability factor, is a potent
angiogenic factor and endothelial cell-specific
mitogen
(8, 9, 10) which is regulated by hypoxia
in vitro(6, 11, 12) and in
vivo(5, 6, 7, 13, 14, 15) .
This hypoxic induction of VEGF is due to an increase in the steady
state level of the mRNA for VEGF
(6) .
Cell Lines and Culture Conditions
PC12 rat
pheochromocytoma cells were the generous gift of Dr. Eva J. Neer
(Brigham and Women's Hospital, Boston, MA). The cells were
routinely grown in Dulbecco's modified Eagle's medium
(Sigma) with 10% fetal bovine serum and used for all experiments at
70% confluence. Cells were cultured under either normoxic
conditions (5% CO
, 20% O
, 75% N
in
a humidified Napco incubator at 37 °C or hypoxic conditions (5%
CO
, 1% O
, 94% N
) in a ESPEC triple
gas incubator (Tabai-Espec Corp., Osaka, Japan).
Cloning and Sequencing of the Rat Genomic VEGF
Gene
3 10
bacteriophage clones from a
Lambda-DASH (Stratagene) 8-week-old rat Sprague-Dawley genomic library
were screened with a cDNA probe corresponding to VEGF
, a
novel VEGF isoform that contains exon 1 spliced to exon 8
(12) .
Secondary and tertiary screening of the genomic clones were performed
using exon 1 (NL23, 5`AACCATGAACTTTCTCTCTT3`) or exon 8 (NL24,
5`GGTGAGAGGTCTAGTTCCCGA3`) oligonucleotides derived from the nucleotide
sequence of rat VEGF cDNA. Distinct VEGF genomic clones hybridizing to
NL23 or NL24 were isolated. Bacteriophage DNA from each 5` or 3`
bacteriophage clone was purified, digested with PstI, and
cloned into a Bluescript vector (Stratagene) generating a PstI
library of the 5` or 3` region of the VEGF gene, respectively. This
PstI library was then screened with
[
-
P]NL23 or
[
-
P]NL24 oligonucleotides. This approach
yielded a 3.0-kb PstI fragment (clone 5.1) and a 2.2-kb
PstI fragment (clone 11.4) from the 5` and 3` regions of the
VEGF gene, respectively. Sequences were obtained 5` to clone 5.1 by
screening an EcoRI library generated from the original
bacteriophage clones as described above using an oligonucleotide from
the most 5` sequence of clone 5.1 to screen the EcoRI library.
A 10-kb EcoRI fragment overlapping with the PstI
fragment from clone 5.1 was obtained by this strategy (clone 5EPst). An
oligonucleotide complementary to the most 5` sequence of clone 5.1 was
used to sequence across the PstI site of clone 5EPst.
Sequences 5` to the PstI site were then used to screen the
original PstI library and yielded a 6.0-kb PstI
fragment that maps immediately 5` to clone 5.1. Sequences were obtained
3` to clone 11.4 by constructing an EcoRI R1 library from the
3` bacteriophage clone and then screening the library with an
oligonucleotide complementary to the 3`-most region of clone 11.4.
Using this strategy, a 3.8-kb EcoRI fragment (clone 11.36) was
identified. Sequencing of these clones was performed by the dideoxy
chain termination method using Sequenase (Stratagene) initially with T3
and T7 primers and subsequently, in a progressive fashion, with
oligonucleotides complementary to the sequences obtained from the
respective clones. Both strands of all clones were sequenced.
Nuclear Runoff Transcription Assay
Nuclei were
obtained and assays were performed by a modification of previously
described procedures
(24) . Briefly, cells were scraped and lysed
in Nonidet P-40 buffer (10 mM Tris, pH 7.5, 10 mM
NaCl, 3 mM MgCl, and 0.5% Nonidet P-40), and
nuclei were resuspended in 175 µl of glycerol storage buffer (40%
glycerol, 10 mM Tris, pH 7.5, 5 mM MgCl
,
80 mM KCl, and 0.1 mM EDTA) and stored in liquid
N
. Eight µl of 100 mM solutions of ATP, CTP,
and GTP, 20 µl of [
-
P]UTP (3000
Ci/mmol, DuPont NEN), and 1 µl of 100 mM dithiothreitol
were added to the nuclear suspension, and the transcription runoff was
allowed to proceed at 30 °C for 30 min. The reaction was terminated
with the addition of DNase I (300 units) and CaCl
(final
concentration of 1 mM) and then incubated at 30 °C for an
additional 10 min. One µl of proteinase K (20 mg/ml) and 25 µl
of SET (5% SDS, 50 mM EDTA, 100 mM Tris, pH 7.5) were
added, and the mixture was incubated for 30 min at 37 °C.
Subsequently, 550 µl of 4 M guanidinium isothiocyanate and
90 µl of 3 M NaOAc were added, and the mixture was
extracted once with phenol/chloroform/isoamyl alcohol (25:24:1). The
aqueous phase was removed, and the RNA was precipitated with 1 volume
of isopropyl alcohol. The pellet was resuspended in 300 µl of
guanidinium isothiocyanate, and the RNA was reprecipitated with 1
volume of isopropyl alcohol. The pellet was washed once with 70%
ethanol, dissolved in 100 µl of TES (10 mM Tris, pH 7.2, 1
mM EDTA, 0.1% SDS), and used directly for hybridization.
Nitrocellulose filters (Schleicher and Schuell) were prepared in a DNA
slot blot apparatus with 5 µg of DNA that was previously denatured
with 0.2 M NaOH. The prehybridization and hybridization
solutions consisted of 50% formamide, 5
SSC (0.15 M
NaCl, 15 mM sodium citrate), 1
Denhardt's
solution, 10% dextran sulfate, 100 µg/ml tRNA, and 0.375% SDS.
Hybridization was carried out at 42 °C for 2-3 days. Filters
were washed at 65 °C first in 2
SSC with 0.1% SDS and then
in 0.2
SSC with 0.1% SDS prior to phosphorimaging and
autoradiographic analysis. Murine actin cDNA cloned into Bluescript was
used to normalize the VEGF hybridization signal. Bluescript vector
without insert was used as a negative control. Rat VEGF gene
transcription was assessed using a 900-bp PstI-SmaI
fragment (nucleotides 990-1855, see Fig. 3) from the
5`-untranslated region of VEGF subcloned into Bluescript. Quantitation
of the nuclear runoff assay was performed using a Molecular Dynamics
PhosphorImager.
Figure 3:
Sequence alignment of rat (top)
and human (bottom) VEGF 5`-flanking and -untranslated regions
as analyzed by the BestFit program (Genetics Computer Group Sequence
Analysis Software Package, Version 7.0). Regions matching
transcriptional control consensus sequences (as identified using the
sequence analysis program MacVector 4.1.3) are indicated as follows:
SP1 (solid line), 5`GGGCGG3` (50, 51); AP-1 (hatched
line), 5`TKAGTCA3` (52, 53); AP-2 (open box),
5`CCSCRGGC3` (54, 55); conserved putative HIF-1 sequence (shaded
box) containing a 6/8-bp match to the Epo HIF-1 site, 5`TACGTGCT3`
(20). The major transcription initiation site as identified by primer
extension analysis is indicated by a bent arrow. The
translation initiation codon (ATG) is circled. Numbering for
the rat VEGF gene is as shown. Numbering for the human VEGF gene is
according to GenBank/EMBL Data Bank accession numbers
M63971-M63978. K = G or T; S = G or C; R = G
or A.
Northern Blot Analysis
Northern blots were
performed as described previously
(12) . Briefly, total RNA was
isolated as described previously
(25) . Electrophoresis of 10
µg of RNA/lane was performed in 1% agarose gels with 2.2 M
formaldehyde and transferred to GeneScreen Plus membranes (DuPont NEN).
After UV cross-linking the RNA to the membrane using a Stratalinker
(Stratagene), the filters were prehybridized in 50% formamide, 2
SSC, 10% dextran sulfate, 1% SDS, and 150 µg/ml sheared
salmon sperm DNA. cDNA or genomic DNA fragments used as probes were
prepared by electroelution onto NA-45 paper (Schleicher and Schuell)
from agarose gels. Fragments were labeled using random primer
methodology (26) (Pharmacia Biotech) to a specific activity of at least
1
10
cpm/µg of DNA. Hybridization was performed
at 42 °C for 16 h. The filters were then washed twice at 65 °C
with 2
SSC and 1% SDS for 20 min each and subsequently washed
twice at 65 °C with 0.2
SSC and 1% SDS for 20 min each.
Hybridization with a
-
P-labeled oligonucleotide
complementary to 18 S rRNA was performed as described previously
(27) and used to normalize for the amount of RNA in each lane.
RNase Protection Assay
Total cellular RNA was
isolated as described above. RNase protection assays utilizing
riboprobes that are capable of distinguishing 4 VEGF isoforms were
performed as described previously
(12) . In order to map the 5`
transcription start site, a PstI-NruI (nucleotides
25-1040, see Fig. 3) fragment from the 5` region was
subcloned into Bluescript. Linearization of the plasmid with
SmaI (nucleotide 887, see Fig. 3) and generation of an
antisense riboprobe with T3 polymerase generated a riboprobe of 184
bases.
Primer Extension Assay
The primer Pext5
(5`CTGCCGCCGCTCAGCTCGCCCC3`) was used to perform primer extension on
RNA from normoxic or hypoxic cells according to published procedures
(28).
Transient Transfection Assay
Plasmids containing
VEGF 5`-flanking sequences subcloned 5` to the luciferase gene were
constructed using the luciferase containing eukaryotic expression
vectors pxp2 and pT109luc
(29) . Plasmid DNA was transfected into
cells by electroporation optimized at 280 V in a custom-made
electroporation apparatus
(30) . In all experiments, the test
plasmid (40 µg) was transfected with 4 10
cells
per cuvette (Bio-Rad). The cells in the cuvette were then split equally
and randomly between two tissue culture plates destined to be incubated
at 1% or 21% O
. After 1 h at 21% O
, the plates
designated for hypoxia were transferred to the hypoxia apparatus and
incubated in parallel with the plates remaining at 21% O
for 14-16 h. Luciferase activity was then assayed according
to the manufacturer's protocol (Analytical Luminescence
Laboratory, San Diego, CA) in a Monolight 2010 luminometer (Analytical
Luminescence Laboratory).
using mouse mammary tumor virus or SV40 promoter sequences
directing the luciferase reporter gene introduced by electroporation
and treated in the manner described above The electroporation and
parallel culture conditions described above allow each pair of plates
to be treated identically up to the point of placing the cells in the
hypoxia apparatus, thus controlling for any difference in cell number
distributed between the two plates or transfection efficiency (plating
efficiency and cell viability did not differ between cells grown at 1%
or 21% oxygen under the culture conditions used). Each test plasmid was
transfected at least 10 separate times.
Nuclear Extract and Electromobility Shift Assay
(EMSA)
Nuclear extracts from hypoxic and normoxic cells were
prepared according to the protocol described by Andrews and Faller
(31). Based on VEGF 5`-flanking sequence (nucleotides 1-959),
complementary oligonucleotides containing a HIF-like binding site and
BamHI and SacI recognition sites on the ends (W28,
5`GATCCACAGTGCATACGTGGGCTTCCACAGAGCTC3` and
5`CTGTGGAAGCCCACGTATGCACTGTG3`) were synthesized, annealed, and cloned
into Bluescript. A construct was also prepared which was identical
except that it contained a mutation in the HIF-like binding site (M28,
5`GATCCACAGTGCATCAATGGGCTTCCACAGAGCTC3` and
5`CTGTGGAAGCCCATTGATGCACTGTG3`). The sequences were verified by DNA
sequencing of both strands. This BamHI-SacI fragment
was labeled with Klenow by filling in the 5` overhang with
[-
P]dCTP. The labeled fragment was then
electrophoresed on a 3% agarose gel containing ethidium bromide,
isolated on NA-45 paper, eluted with 1 M NaCl, and
ethanol-precipitated. Ten thousand cpm (
0.2 ng) were used for each
binding reaction. Binding assays were performed according to Semenza
et al.(19) , using calf thymus DNA as a nonspecific
competitor. Binding reactions contained 5 µg of nuclear extract,
0.1 µg of denatured calf thymus DNA (Sigma), 10 mM Tris,
pH 7.5, 50 mM KCl, 50 mM NaCl, 1 mM
MgCl
, 1 mM EDTA, 5 mM dithiothreitol, and
5% glycerol. The reactions were preincubated at room temperature with
or without specific competitors for 5 min before the radiolabeled probe
was added. Incubation was then continued for an additional 10 min at
room temperature. Reaction products were electrophoresed at 4 °C in
a 5% nondenaturing polyacrylamide gel with 0.3
TBE (30
mM Tris, 30 mM boric acid, 0.06 mM EDTA, pH
7.3 at 20 °C). Competitor DNA was prepared by mixing equimolar
quantities of the complementary oligonucleotides and was used at an
100-fold molar excess relative to the radioactive probe. Epo18
oligonucleotides
(22) were 5`GCCCTACGTGCTGCCCTCG3` and
5`CGAGGGCAGCACGTAGGGC3` and were the generous gift of Drs. Eric Huang
and H. Franklin Bunn (Brigham and Women's Hospital, Boston, MA).
The AP-1 consensus oligonucleotides 5`CTAGTGAT-GAGTCAGCCGGATC3` and
5`GATCCGGCTGACTCATCACTAG3` were obtained from Stratagene and were used
as a nonspecific competitor.
VEGF Steady State mRNA Is Up-regulated by Hypoxia in
PC12 Cells
VEGF mRNA has previously been demonstrated to be
up-regulated in response to decreased O tension
(6) in all cell types and cell lines examined with the exception
of endothelial cells
(32) . The rat pheochromocytoma tumor cell
line PC12 has previously been demonstrated to induce VEGF mRNA in
response to a variety of activated second messenger
systems
(33) . Fig. 1demonstrates that VEGF mRNA is also
up-regulated by hypoxia in the PC12 cells. After being grown for 6 h in
1% O
, the mRNA for VEGF is increased 12.0 ± 0.6-fold
as determined by RNase protection assay. Analogous to primary cardiac
myocytes
(12) , all four isoforms of VEGF mRNA are induced
equivalently by hypoxia in PC12 cells as determined by RNase protection
assay using isoform specific probes (data not shown).
Figure 1:
RNase
protection assay of hypoxic PC12 cells. Total cellular RNA from PC12
cells grown in 1% or 21% O for 6 h was isolated, and RNase
protection assays were performed with isoform specific riboprobes (12).
The band corresponding to VEGF165 (the VEGF mRNA encoding the
165-amino-acid isoform) is shown with U3 snRNA serving to normalize for
differences in sample loading. Quantitation of each isoform was
performed by cutting the bands directly from a gel and counting them in
a liquid scintillation counter.
Hypoxia Increases the Transcription Rate for
VEGF
PC12 cells were grown at 1% or 21% oxygen tension for 6 h
before nuclei were harvested for nuclear runoff assays as described
under ``Materials and Methods.'' Murine -actin, whose
transcription rate has been previously demonstrated to be minimally
changed by hypoxia
(34) , was used to normalize the hybridization
signal for each runoff assay. The transcription rate for VEGF was
observed to increase 3.1 ± 0.6-fold (n = 7),
p < 0.001 (Fig. 2), in cells grown under hypoxic
conditions for 6 h compared to cells grown at 21% O
.
Similar results were obtained when nuclei were harvested between 3 and
12 h.
Figure 2:
Nuclear runoff transcription assay. Five
µg of denatured rat VEGF plasmid (clone 5.1), murine -actin,
and the plasmid vector alone (Bluescript (BS)) were each bound
to nitrocellulose and were hybridized with
P-labeled
runoff transcripts from nuclei isolated from PC12 cells after 6 h of
hypoxia or normoxia. Results were quantitated using a Molecular
Dynamics PhosphorImager. For each of 7 independent nuclear runoff
assays, a VEGF/actin ratio was calculated separately for hypoxia and
normoxia. The -fold increase in the transcription rate for each of the
7 experiments was then calculated by dividing the hypoxic VEGF/actin
ratio by the normoxic VEGF/actin ratio. The mean ± S.E. increase
in transcription rate for VEGF under hypoxic conditions for all 7
experiments was 3.1 ± 0.6.
Nucleotide Sequence of the Rat VEGF Gene 5`
Region
Rat VEGF genomic DNA was cloned from a bacteriophage
library and sequenced as described under ``Materials and
Methods.'' The nucleotide sequence of the 5` region of the rat
VEGF gene, including 1 kb of 5`-untranslated and
1 kb of
5`-flanking sequence, is shown in Fig. 3aligned to the human
VEGF sequence
(35) . The overall homology in the 5`-flanking and
-untranslated region was greater than 79%. Ten potential HIF-1 sites
were identified with a 6/8- or 7/8-bp match to the Epo HIF-1 site in
the 5`-flanking region. None of the potential HIF-1 sites conforms to
the consensus sequence recently proposed by Semenza et
al.(21) . The position of only one of these possible HIF-1
sites (5`TACGTGGG3`, nucleotides 65-72, Fig. 3) is
conserved between the rat and human genes and is located within a 28-bp
region that is 100% conserved between rat and human VEGF. A 5-bp
element, 5`CACAG3`, is present in the Epo enhancer element and is
located 4 nucleotides 3` to the HIF-1 site. This element, which is
absolutely required for hypoxic induction of Epo
(21) , is also
present 4 nucleotides 3` to the conserved putative HIF-1 site in the
VEGF 5`-flanking region. A single consensus AP-1 site is conserved and
is found 28 bp 3` to the putative HIF-1 site. Three AP-2 sites are
present in the VEGF 5`-UTR and -flanking region (nucleotides 880, 890,
and 1842, see Fig. 3), but only one of these is conserved in the
human gene. In addition, a cluster of conserved SP1 sites is found in
the minimal promoter (nucleotides 888-965, see Fig. 3) as
defined by primer extension and transient expression assays (see
below), while no TATA or CAAT box is found in either the rat or the
human
(35) promoter sequences.
Mapping the Transcription Initiation Site
The
transcription initiation site was mapped by primer extension
(Fig. 4A) and by the RNase protection assay
(Fig. 4B) as described under ``Materials and
Methods.'' Both analyses yielded similar results. The initiation
site maps to the same region as described previously for the human gene
(Fig. 3) and does not appear to change with hypoxia. While a
single predominant transcription initiation site is evident using both
methods, several other minor potential transcription initiation sites
within 5-10 nucleotides of the most abundant start site were
identified using the RNase protection assay.
Figure 4:
Mapping
of the VEGF transcription start site in PC12 cells. A, primer
extension analysis of hypoxic and normoxic VEGF mRNA. Primer extension
products generated using oligonucleotide Pext5 were electrophoresed
alongside a DNA sequencing ladder primed by Pext5. The location of the
major primer extension product is indicated by an arrow, and
the C nucleotide (nucleotide 964) complementary to the transcription
start site is indicated by an asterisk. B, RNase
protection assay. A 184-bp riboprobe was generated, and hybridization
was performed as described under ``Materials and Methods.''
P, undigested probe; N, normoxic PC12 mRNA;
H, hypoxic PC12 mRNA; t, tRNA. The most prominent
band is 83 bp in size based on a DNA sequencing ladder present in
adjacent lanes. RNA has a faster mobility through the gel than DNA of
the same length. When DNA markers are used to estimate the size of a
protected RNA fragment, the correct size is 5-10% smaller than
this estimate (56). Therefore, the transcription start site by RNase
protection assay is mapped 75-79 bp 5` to the NruI site
(nucleotides 964-968, see Fig. 3).
Transient Expression Reporter Assays
To locate the
cis-regulatory sequences responsible for the transcriptional
activation of VEGF, a series of constructs containing 5`-flanking
sequences placed 5` to the promoterless luciferase gene (pxp2) were
transfected into PC12 cells by electroporation. Luciferase activity was
assayed 13-15 h later. All constructs gave approximately 200-fold
more light units in the luciferase assay than with the vector alone
when transfected into PC12 cells and grown in 21% O.
. No change was seen in the hypoxic induction of
luciferase activity when 3`-flanking or 3`-untranslated region
sequences were placed 5` to the VEGF minimal promoter or 5` to the
thymidine kinase promoter (pT109luc) (data not shown). The most
significant decrement in hypoxia-inducible luciferase activity occurred
after deletion of a PvuII-SacI fragment (nucleotides
15-230, Fig. 3) from the 5` end of the
PvuII-PstI construct (Fig. 5A). The
enhancer function of this fragment was confirmed by demonstrating that
it could confer hypoxic responsiveness in either orientation when
placed 5` to a thymidine kinase luciferase construct (pT109luc or
pT81luc) (Fig. 5B). Notably, this 215-bp fragment
contained a region of highly conserved sequence between the rat and
human genes (40/43 or 93%). Within this region of conserved sequence
homology, a potential HIF-1 site was identified. Oligonucleotides were
synthesized corresponding to this putative HIF-1 site (W28) as well as
a 3-base pair substitution in the site (M28) that had previously been
shown to eliminate HIF-1 activity
(36) both functionally in
reporter assays and in EMSA assays. The synthetic putative HIF-1 site
(W28) placed 5` to the VEGF minimal promoter in pxp2 conferred a
statistically significant increase in the hypoxia/normoxia ratio of
luciferase activity in the reporter assay, while no significant change
was seen with M28, the oligonucleotide designed to eliminate HIF-1
activity (Fig. 5A).
Figure 5:
Determination of hypoxia responsive
elements in the 5`-flanking region of the rat VEGF gene. PC12 cells
were transfected with luciferase expression vectors containing variable
fragments of the 5`-flanking region (as depicted in A and
B) and exposed to hypoxia for 13-15 h. Luciferase
activity is expressed as the ratio of activity in hypoxic (1%
O) to normoxic (21% O
) cells. The standard
error of the mean is given for at least 10 different transfections. The
transcription initiation site is indicated by a bent arrow.
Nucleotide numbers (taken from Fig. 3) for the following restriction
sites are: PvuII (23), SacI (239), MluI
(523), SmaI (885), PstI (986). The EcoRV
site maps approximately 700 nucleotides 5` to the PvuII site.
A, constructs and results of transfection of plasmids
containing 5`-flanking fragments in pxp2. B, constructs and
results of transfection of plasmids containing 5`-flanking fragments in
pT109luc.
Responses with these luciferase
constructs were not restricted to PC12 cells. An increase in luciferase
activity was observed in transfected hypoxic primary rat neonatal
cardiac myocytes relative to transfected normoxic myocytes (data not
shown).
Binding of Hypoxia-inducible Factors to an
Oxygen-regulated Element within the VEGF Enhancer
The 28-bp
element (W28) shown to confer increased hypoxia responsiveness to the
minimal promoter was assessed for its ability to bind hypoxia-inducible
factors (Fig. 6). In electromobility shift assays, a specific,
hypoxia-inducible, DNA band shift was observed that was competitively
blocked with an excess of either unlabeled oligonucleotide W28 or an
oligonucleotide (E18) containing a HIF-1 binding site from the Epo
hypoxia responsive enhancer. Conversely, the mutant oligonucleotide M28
was unable to competitively inhibit the W28 band shift, nor was it able
to specifically retard a hypoxia-inducible species in the EMSA assay.
As described previously when EMSA is performed with hypoxic nuclear
extracts using E18 as probe
(20) , a constitutively expressed
sequence-specific DNA binding activity, and a nonspecific DNA binding
activity, were also detected in the PC12 nuclear extracts using W28 as
probe. The constitutive species could be competitively blocked with an
excess of unlabeled W28, E18, or AP1 oligonucleotide, but was only
partially competed with M28. Additionally, mutant oligonucleotide M28
was unable to specifically shift the constitutive species in the EMSA
assay. The nonspecific DNA complex was competitively inhibited by all
competitors used.
Figure 6:
Electrophoretic mobility shift assay
(EMSA). The position of hypoxia-inducible complexes (HI),
constitutive complexes (C), nonspecific complexes
(NS), and free probe (FP) are indicated. Labeled
oligonucleotide probes and unlabeled oligonucleotides used as
competitors in 100-fold molar excess are indicated. WT,
M, AP1, and EPO are the W28, M28, AP-1, and
E18 oligonucleotides, respectively, as described under ``Materials
and Methods.'' Nuclear extract is either from hypoxic (1%
O) or normoxic (21% O
) PC12
cells.
Identification and Sequence of the 3`-Untranslated Region
of VEGF mRNA
VEGF was cloned from a bacteriophage library and
sequenced as described under ``Materials and Methods.'' Clone
11.4 was demonstrated to begin 6 nucleotides 3` to the translation
termination codon of the VEGF protein based on the VEGF cDNA
sequence
(37) . The nucleotide sequence of clone 11.4 is given in
Fig. 7
. Four potential polyadenylation signals
(38) are
identified in this sequence (nucleotides 364, 1193, 1211, and 1852) as
well as multiple AUUUA and polypyrimidine sequence motifs
(Fig. 7) that have been demonstrated to mediate changes in RNA
stability
(39, 40, 41) .
Figure 7:
Analysis of the 3` region of the rat VEGF
gene. The nucleotide sequence of genomic clone 11.4, as described under
``Materials and Methods,'' begins 6 bp 3` to the VEGF
translation termination codon (37). The regions containing potential
regulatory sequences are indicated as follows: AUUUA (open
bars), polypyrimidine tract (hatched bars; defined as
greater than 28 bp with pyrimidine content greater than 85%), and
polyadenylation signals (solid bars)
(38).
Northern blot
analysis was performed in order to map the approximate transcription
termination site. As demonstrated in Fig. 8, A and
B, probes derived from the 5`-UTR, VEGF coding region, or
approximately 2 kb of 3`-flanking region hybridized to a 3.7-kb mRNA
species that is increased in hypoxic cells. Probe F, located greater
than 2 kb downstream from the translational termination codon, gave no
hybridization signal. An additional 800 bp 3` to this fourth
polyadenylation site has been sequenced without evidence of any further
polyadenylation sites (data not shown). These results are consistent
with the frequent use of the fourth polyadenylation site as a site for
transcription termination.
Figure 8:
Mapping
the transcription termination site of the rat VEGF gene. A,
restriction map of the 3` region of the VEGF gene and schematic
representation of probes A and D-F used for
Northern blot analysis. The nucleotide numbers for the following
restriction enzyme sites (taken from Fig. 7) are as follows (listed in
order from 5` to 3`): PstI (1), BamHI (760),
EcoRI (1630), EcoRI (1860), PstI (2210). The
most 3` PstI site is approximately 500 nucleotides downstream
of PstI (2210).
A-A
, consensus
polyadenylation sequences (38). Probe B was a PstI
(86)-SmaI (1857) fragment from the 5`-flanking and 5`-UTR
sequence (see Fig. 3). Probe C was a 1-kb cDNA fragment
encoding mouse VEGF (33). B, Northern blot analysis of total
RNA from PC12 cells cultured under 1% or 21% O
. Probes are
as described in A. Each of the probes was made to an
approximately equal specific activity. After washing the blots,
panel F was exposed to autoradiography at -70 °C for
4 days. Panels A-E were exposed for 1 day or less. 18 S
oligonucleotide hybridization signals are shown as a control to
demonstrate the presence of RNA in all lanes. Based on the presence or
absence of a hybridization signal, the transcription termination site
is mapped between probe E and probe
F.
(
)
suggest that hypoxia increases the half-life of VEGF mRNA.
Further evidence for a post-transcriptional mechanism regulating the
hypoxic induction of VEGF comes from the use of the protein synthesis
inhibitor cycloheximide. Cycloheximide results in an increase in the
steady state mRNA level under normoxic conditions
(6) and
markedly increases the half-life of VEGF mRNA in Hep3B cells switched
from an atmosphere containing 1% O
to one containing 21%
O
(11) . The cis-regulatory elements that
mediate this change in VEGF mRNA stability by hypoxia are likely to lie
in the 3`-UTR of the VEGF gene and remain to be characterized.
3.7 kb in length, in good
agreement with Northern blot results.
/EMBL Data Bank with accession number(s) U22372 and
U22373.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.