(Received for publication, March 13, 1995; and in revised form, June 13, 1995)
From the e 1, 61231 Bad Nauheim,
Germany
Vascular endothelial growth factor (VEGF) is an endothelial specific angiogenic mitogen secreted from various cell types including tumor cells. Increasing evidence suggests that VEGF is a major regulator of physiological and pathological angiogenesis, and the VEGF/VEGF receptor system has been shown to be necessary for glioma angiogenesis. Hypoxia seems to play a critical role in the induction of VEGF expression during glioma progression. C6 glioma cells provide an in vivo glioma model for the study of tumor angiogenesis, and the expression of VEGF in C6 cells has been shown to be up-regulated by hypoxia in vitro. However, little is known about the molecular mechanism of hypoxic induction of VEGF. Here, we demonstrate that hypoxic induction of VEGF in C6 cells is due to both transcriptional activation and increased stability of mRNA. Nuclear run-on assays revealed a fast and lasting transcriptional activation, whereas the determination of mRNA half-life showed a slower increase of mRNA stability during hypoxia. Reporter gene studies revealed that hypoxia responsive transcription-activating elements were present in the 5`-flanking region of the VEGF gene. These results suggested that several distinct molecular mechanisms were involved in hypoxia-induced gene expression and were activated in a biphasic manner.
Normal embryonic development, wound healing, and certain
physiological processes such as corpus luteum formation require the
formation of new blood vessels from pre-existing vessels, a process
termed angiogenesis(1) . Angiogenesis also plays an important
role in some pathological events, for example, in solid tumor growth,
diabetic retinopathy, or rheumatoid arthritis(2) . The growth
of solid tumors is angiogenesis-dependent(2) . Several
polypeptide growth factors such as fibroblast growth factors, vascular
endothelial growth factor (VEGF), ()transforming growth
factor-
are thought to be involved, directly or indirectly, in the
regulation of angiogenesis(2, 3) . Among these
factors, VEGF has been reported to be an endothelial cell-specific
mitogen(4, 5, 6) . In situ hybridization studies showed that the expression of genes encoding
VEGF and its receptors correlated both spatially and temporally with
brain angiogenesis during embryonic
development(7, 8) . (
)Also in glioma, in
which angiogenesis is one of the hallmarks of malignancy, the higher
grade malignant astrocytoma showed a higher expression of VEGF mRNA in
tumor cells as well as VEGF receptor mRNA in endothelial
cells(9) . In vivo studies revealed that growth of
solid tumors could be inhibited by injection of a specific antibody
capable of blocking VEGF-induced angiogenesis (10) or by
overexpressing a dominant-negative VEGF receptor mutant(11) .
These observations suggested that VEGF was a key regulator of both
embryonic and tumor angiogenesis. In glioblastoma multiforme, the
highest expression of the VEGF gene was observed in palisading cells
surrounding necrotic areas(9, 12) , which indicated
that hypoxia might up-regulate VEGF gene expression in vivo.
It has been shown that VEGF gene expression is up-regulated by hypoxia in vitro and in
vivo(12, 13, 14, 15) . Taking
these observations together, it is conceivable that focal hypoxia is
working as an essential trigger for pathological angiogenesis through
up-regulation of VEGF gene expression. To define the physiological and
pathological roles of hypoxia in angiogenesis in vivo, it is
important to understand the molecular mechanisms by which hypoxia
up-regulates VEGF gene expression in vitro.
In addition to VEGF, hypoxia is known to up-regulate the expression of several genes including the erythropoietin (EPO)(16) , tyrosine hydroxylase(17) , platelet-derived growth factor-B chain(18) , phosphoglycerate kinase 1, and lactate dehydrogenase A genes(19) . Among these molecules, EPO has been most extensively studied. A sequence in the 3`-flanking region of the EPO gene has been identified as a hypoxia-responsive enhancer in liver, and a hypoxia-inducible factor 1 has been found to bind this enhancer sequence(20) .
Although several similarities between the
gene expression of VEGF and EPO have been reported, including
CoCl-induced expression (14) , little is known
about the molecular mechanisms of the regulation of VEGF gene
expression by hypoxia. It has been shown that hypoxia-induced synthesis
of VEGF is regulated at the RNA level in cultured cells, including C6
glioma cells(12, 13, 14) , cardiac myocytes (21) , and vascular smooth muscle cells(22) . In this
study, we used C6 glioma cells, which provide an in vivo glioma model(13) , to analyze the molecular mechanisms
which mediate VEGF mRNA induction by hypoxia. We present evidence that
the hypoxia-induced increase of VEGF gene expression is due to both
transcriptional activation and increased mRNA stability. Furthermore,
the timing of the transcriptional activation is different to that of
the increase in mRNA stability during the course of the hypoxic
response, suggesting that several distinct molecular mechanisms are
involved in the cellular response to hypoxia. In addition, we
demonstrate that the 5`-flanking region of the VEGF gene confers the
transcriptional activation in C6 cells in response to hypoxia.
In vitro transcription and isolation of the resulting RNA were performed as
described by Birch and Schreiber (26) with modifications as
described below. The nuclei (5 10
in 50 µl of
glycerol storage buffer) were thawed and mixed with 50 µl of 2
reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl
, 300 mM KCl, 10 mM
dithiothreitol, 400 units/ml placental ribonuclease inhibitor, 20
mM creatine phosphate, 200 µg/ml creatine phosphokinase)
and 1 mM each of ATP, CTP, and GTP, and 100 µCi of
[
-
P]UTP (3000 Ci/mmol). The reaction was
incubated at 25 °C for 30 min with shaking, 20 units of DNase I
(RNase-free) were added, and the reaction mixture was incubated at 25
°C for further 5 min. Proteinase K and SDS were added to a final
concentration of 150 µg/ml and 0.5%, respectively, and incubation
continued at 37 °C for 30 min. The reaction was extracted with an
equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v), and
RNA was precipitated from the aqueous phase with ethanol in the
presence of 2.5 M ammonium acetate. The precipitate was
resuspended in 200 µl of 5 mM MgCl
, treated
with 100 units of DNase I (RNase-free) at 37 °C for 60 min, and
then EDTA and SDS were added to a final concentration of 15 mM and 0.5%, respectively. Following extraction with an equal volume
of phenol/chloroform/isoamyl alcohol and ethanol precipitation, the
precipitate was suspended in 100 µl of 10 mM Tris-HCl, pH
8.0, 1 mM EDTA. Parallel samples were processed in the absence
or presence of 1 µg/ml
-amanitin.
For hybridization of the
radiolabeled RNA to specific DNA targets, linearized plasmid DNAs (15
µg, per slot) were immobilized onto a nylon membrane (GeneScreen
Plus; DuPont NEN) using a Bio-Dot SF microfiltration apparatus
(Bio-Rad). The immobilized plasmid DNAs were as follows: murine
VEGF cDNA(7) , chick
-actin
cDNA(24) , both cloned in pBluescript KS (Stratagene), and
pBluescript KS DNA to assay for nonspecific hybridization. The filters
were prehybridized overnight at 42 °C in 1.5 ml of buffer
containing 20 mM Pipes, pH 6.4, 50% formamide, 2 mM EDTA, 0.8 M NaCl, 0.2% SDS, 1
Denhardt's
solution (0.02% Ficoll, 0.02% bovine serum albumin, 0.02%
polyvinylpyrrolidone), 200 µg/ml E. coli tRNA
(RNase-free), 100 µg/ml poly(A). Hybridization was at 42 °C for
48 h in the same solution supplemented with 4.5
10
cpm of
P-labeled RNA. The filters were washed twice
in 2
SSC, 0.5% SDS at 42 °C for 30 min, twice in 0.3
SSC, 0.5% SDS at 42 °C for 30 min, and then incubated with
10 µg/ml RNase A in 2
SSC at 37 °C for 30 min. Filters
were washed in 2
SSC at 37 °C for 30 min, and finally in
0.3
SSC at 37 °C for 30 min. The amount of
P-labeled RNA hybridized to each plasmid DNA on the
filters was measured using a PhosphorImager. The amount of VEGF mRNA
was standardized by comparison with the amount of
-actin mRNA.
After measurement, the filters were exposed to Kodak XAR-5 film with
intensifying screens at -80 °C.
Figure 4: Deletion constructs containing regions from the human VEGF gene tested for responsiveness to hypoxia. A, diagram of the 5`-flanking and 5`-untranslated regions of the human VEGF gene. Nucleotides are numbered from the translation start site, and the transcription start site is indicated with an arrow(-1038). B, VEGF-luciferase deletion constructs and degrees of induction by hypoxia. These constructs were transfected into C6 cells, and the degree of induction by hypoxia was determined. Mean and S.E. of induction were obtained from three independent experiments. Restriction sites are: Kpn, KpnI; Spe, SpeI; Sac, SacI; Ban, BanI; Pst, PstI; Apa, ApaI; Nhe, NheI; Nar, NarI.
Plasmids were transfected into C6 cells by Lipofectin Reagent (Life
Technologies, Inc.) essentially following the protocol from the
manufacturer. C6 cells were seeded (3 10
cells/60
mm-diameter culture plate) 24 h before transfection. Cells were
transfected with 5 µg of test plasmid and 5 µg of control
plasmid. For a given test plasmid, 2 plates were transfected at the
same time. The cells were incubated with DNA/Lipofectin mixture for 6
h, and then the mixture was replaced by normal growth medium. Following
24 h under normoxia, one of the two plates was incubated for 18 h under
normoxia, the other for 18 h under hypoxia, respectively. Cell extracts
were prepared using Reporter Lysis Buffer (Promega), and measured for
luciferase and
-galactosidase activity. Luciferase activity was
measured by AutoLumat LB 953 (Berthold) with Luciferase Assay System
(Promega), and
-galactosidase activity was measured by using
chlorophenol red-
-D-galactopyranoside as
substrate(30) . For standardization of transfection efficiency,
luciferase activity was divided by
-galactosidase activity
(luciferase/
-galactosidase). The degree of induction by hypoxia
for each test plasmid was then determined as the ratio of standardized
luciferase activity in cells under hypoxia to that in cells under
normoxia.
Figure 1:
Hypoxic induction
of VEGF mRNA in C6 cells. C6 cells were incubated either under normoxia
or under hypoxia (1% O) for the number of hours indicated.
Total RNA (20 µg/lane) was analyzed by Northern analysis using
probes for VEGF (top panel) and
-actin (bottom
panel).
Figure 2:
Nuclear run-on analysis of transcription
of the VEGF and -actin genes in C6 cells during incubation under
hypoxia. Nuclei were prepared from C6 cells under normoxia or after
hypoxic incubation for 3 or 15 h. After in vitro transcription
in the presence of [
-
P]UTP,
P-labeled RNA (4.5
10
cpm) was
hybridized to VEGF cDNA,
-actin cDNA, and pBluescript plasmid
immobilized on a nylon membrane.
Figure 3:
VEGF mRNA stability under normoxia or
hypoxia. A-C, autoradiographs of Northern analyses. C6 cells
cultured under normoxia (A) or after hypoxic incubation for 3 (B) or 15 (C) h were treated with actinomycin D (ActD), and then total RNA samples were collected every hour
and analyzed by Northern analysis for VEGF mRNA and -actin mRNA.
Autoradiography for VEGF mRNA was at -80 °C for 14 days for
the normoxia filter (A) and 7 days for the filters for both
hypoxia 3 (B) and 15 (C) h. D, quantitative
analysis of VEGF mRNA stability from two independent experiments.
Quantitative analysis was performed with a PhosphorImager. Comparison
of VEGF mRNA levels in the cells at each time point was achieved by
normalization to
-actin mRNA.
Angiogenesis is one of the most important aspects in various kinds of physiological and pathological processes(1, 2) . For example, a solid tumor could not grow beyond a certain size without angiogenesis (2) . Clarification of the molecular mechanisms regulating angiogenesis is of great benefit, because it might lead to the establishment of new tumor therapy based on the suppression of neovascularization. Recently, we and others have demonstrated the important role of VEGF in tumor angiogenesis(9, 10, 11) . In particular, increased synthesis of VEGF in response to focal hypoxia seems to be critical(9, 12) . Astrocytomas are tumors derived from astroglia. Astrocytomas are known to progress from low malignant to highly malignant tumors (glioblastoma multiforme) during the course of patients' history, and angiogenesis seems to play an important role in this progression. C6 glioma cells provide a good in vivo glioma model when they are transplanted into syngeneic rats(13) . The expression patterns of VEGF and its receptors in this model have been shown to be indistinguishable from glioblastomas in patients(13) , which suggests that the same mechanisms as in human tumors are involved in this animal model. Hypoxia has been shown to up-regulate the expression of the VEGF gene in C6 cells (12, 13, 14) . With this C6 glioma model, we have investigated the molecular mechanisms underlying tumor angiogenesis, focusing on hypoxia-induced expression of VEGF.
Hypoxia-induced expression of VEGF in C6 cells in vitro was regulated at the RNA level. The level of VEGF mRNA had already increased (3-4-fold) after 3 h of incubation under hypoxia, reached the steady-state level (8-10-fold) around 15 h of incubation, and remained elevated during the period examined (24 h). To elucidate the mechanisms which contribute to the increase of VEGF mRNA under hypoxia, we performed nuclear run-on assays. Nuclear run-on assays revealed that the transcriptional rate of the VEGF gene was already up-regulated after 3 h of incubation under hypoxia (3.4 ± 0.5-fold) and persisted after 15 h of incubation (2.5 ± 0.6-fold). This result indicated that the early induction of VEGF mRNA synthesis was mediated by transcriptional activation. However, VEGF mRNA levels after 15 h of hypoxic incubation (around 8-10-fold higher than normoxic levels) could not be accounted for by transcriptional activation only. Therefore, we measured half-lives of VEGF mRNA in C6 cells under normoxia and after hypoxic treatment for 3 and 15 h. Interestingly, the half-life of VEGF mRNA after 15 h of hypoxic treatment was found to be prolonged (about 2.6-fold compared to normoxic conditions), whereas 3 h of hypoxic treatment had no significant effect on the half-life of VEGF mRNA. These results revealed that hypoxia-induction of VEGF mRNA synthesis after 15 h of hypoxia is mediated not only by transcriptional activation but also by the increased stability of mRNA. It is also noteworthy that, in contrast to the fast transcriptional activation, stability of mRNA increased later during the time course under hypoxia. This demonstrates the involvement of several distinct molecular mechanisms in the hypoxic induction of VEGF. The regulation of the VEGF gene by hypoxia appears to be similar to the regulation of the tyrosine hydroxylase gene expression in PC12 cells(17, 31) . Hypoxia increased the tyrosine hydroxylase mRNA level in PC12 cells through both transcriptional activation and increased stability of mRNA. In these experiments, the time courses of transcriptional activation and increased stability of tyrosine hydroxylase mRNA under hypoxia were also found to be different. As compared with the fast activation of transcriptional rate, the stability of tyrosine hydroxylase mRNA increased much slower during the time course of hypoxic incubation, which is consistent with our results for hypoxia-induced VEGF gene expression. These observations suggest that general responsive mechanisms to hypoxia exist which are temporally precisely regulated and common to different cell types and the expression of different genes.
Several similarities between the hypoxia-induced gene
expression of VEGF and EPO have been reported(14) . For
example, the expression of both genes was induced by CoCl treatment, or the hypoxia-induced expression was suppressed in
the presence of carbon monoxide(14, 32) . (
)These observations suggest that a heme protein works as
the oxygen sensor to regulate EPO and VEGF gene expression, for
instance, in the soil bacterium Rhizobium
meliloti(33, 34) . Furthermore, it has been
discussed that hypoxia-inducible factor 1, which is generally activated
by hypoxia and binds the EPO hypoxia responsive enhancer sequence in
various types of cells, might be involved in hypoxia-induced expression
of VEGF. On the other hand, protein kinase C activation led to the
increased expression of the VEGF gene(28, 35) ,
whereas it inhibited the hypoxia-induced expression of
EPO(36) . This observation implies that different signaling
pathways might be involved in the regulation of VEGF and EPO genes
expression. In addition, mutation of p53, which was associated with
astrocytoma progression (37) , could potentiate protein kinase
C induction of VEGF(38) . Involvement of cAMP-dependent protein
kinase pathway in VEGF gene expression was also indicated(35) .
These observations demonstrate the complexity of the molecular
mechanism involved in VEGF gene regulation, including hypoxia-induced
expression. Our data, in which transcriptional activation and increased
stability of mRNA are differentially regulated during the time course,
may support this notion. It might be possible that the cells exposed to
hypoxic conditions first respond to decreased oxygen tension through
transcriptional activation and then secondary stimuli such as metabolic
changes give rise to the increased stability of mRNA via different
pathways. To prove this hypothesis, we need to clarify in more detail
the molecular mechanisms involved in this response, including
cis-acting elements and trans-acting factors which are involved in VEGF
gene induction and mRNA stabilization by hypoxia.
We tested the 5`-flanking region of the human VEGF gene (28) for hypoxia responsive elements by transient transfection with a series of deletion constructs. These studies demonstrated that a 293-bp SacI-BanI fragment (-2218 to -1926) in the 5`-flanking region contains hypoxia responsive elements. Transcriptional activation of the VEGF gene in C6 cells by hypoxia is thought to be, in part, conferred by this genomic fragment. It contains one GC box sequence (-2138 to -2133) which is a potential binding site for the transcription factor Sp1(28) . Recently, it was reported that a 100-bp fragment (-1882 to -1782) from the 5`-flanking region of the human VEGF gene contains a hypoxia responsive element which is functional in HeLa cells(39) . This 100-bp fragment (-1882 to -1782) is located near the 5` end of the BanI-NheI fragment. In our study, this BanI-NheI fragment does not confer hypoxia responsiveness to C6 cells. One possible explanation for this difference is that the regions of the VEGF gene which confer hypoxic responsiveness differ between cell types. Another explanation is that several distinct regions are involved in hypoxia-induced transcriptional activation of the VEGF gene. Further studies are necessary to clarify which pathological conditions caused by hypoxia are able to activate these cis-acting elements; for example, decreased oxygen tension, metabolic changes, or accumulation of metabolites. In this study, a cis-acting element of the VEGF gene was identified which confers hypoxic responsiveness to C6 glioma cells. This SacI-BanI fragment will be useful in elucidating the molecular mechanisms underlying tumor angiogenesis during the progression of glioma.