©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hypoxia-induced Transcriptional Activation and Increased mRNA Stability of Vascular Endothelial Growth Factor in C6 Glioma Cells (*)

(Received for publication, March 13, 1995; and in revised form, June 13, 1995)

Eiji Ikeda Marc G. Achen (§) Georg Breier Werner Risau (¶)

From the Max-Planck-Institut für physiologische und klinische Forschung, W. G. Kerckhoff-Institut, Abteilung Molekulare Zellbiologie, Parkstrabetae 1, 61231 Bad Nauheim, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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), (^1)transforming growth factor-beta 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) . (^2)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(2)-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.


EXPERIMENTAL PROCEDURES

Chemicals

[alpha-P]dCTP (3000 Ci/mmol), [alpha-P]UTP (3000 Ci/mmol), and human placental ribonuclease inhibitor were purchased from Amersham. Ribonucleotide triphosphates were from Pharmacia Biotech Inc. Creatine phosphate, creatine phosphokinase, tRNA from bakers' yeast, tRNA from Escherichia coli (RNase-free), DNase I (RNase-free), proteinase K, alpha-amanitin, poly(A), RNase A, actinomycin D, and chlorophenol red-beta-D-galactopyranoside were from Boehringer Mannheim. Formamide was from Fluka.

Cell Line and Culture Conditions

Rat C6 glioma cells were obtained from the American Type Culture Collection. C6 cells were cultured in Dulbecco's modified Eagle's medium (4500 mg/liter glucose, Life Technologies, Inc.) containing 10% fetal bovine serum (Sigma), and incubated at a CO(2) level of 5% either with 20% O(2) (atmospheric air) for normoxia or with 1% O(2) balanced with N(2) for hypoxia. Hypoxia was generated in an oxygen-regulated incubator (Forma Scientific/Labotect, Model 3015), which took approximately 15 min to equilibrate to 1% O(2). C6 cells were seeded onto culture plates 2 days before hypoxic incubation, and the medium was replaced 1 day before the incubation. The cells were subconfluent at the beginning of hypoxic incubation.

RNA Extraction and Northern Analysis

Total cellular RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction(23) . Equal amounts of total RNA (20 µg/lane) were electrophoresed in 1% agarose gels containing 2.2 M formaldehyde. RNA was blotted by capillary transfer to a nylon membrane (Hybond N; Amersham) in 20 SSC. Filters were cross-linked with UV light (0.5 J/cm^2), and prehybridized for 2-4 h at 42 °C in 50% formamide, 5 SSC (750 mM NaCl, 75 mM NaCitrate, pH 7), 5 Denhardt's solution (0.1% Ficoll, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone), 1% SDS, and 100 µg/ml yeast tRNA. Hybridization was carried out overnight at 42 °C with [alpha-P]dCTP-labeled murine VEGF cDNA (7) . Filters were washed in 2 SSC for 30 min at 42 °C, in 2 SSC, 0.5% SDS for 30 min at 42 °C, in 0.3 SSC, 0.5% SSC twice for 30 min at 42 °C, and then exposed to Kodak XAR-5 film with intensifying screens at -80 °C. Subsequently, filters were hybridized with [alpha-P]dCTP-labeled chick beta-actin cDNA (24) for standardization. Quantitative analysis was also performed with a PhosphorImager SF (Molecular Dynamics).

Nuclear Run-on Assay

C6 cells were treated as follows: (i) normoxia; (ii) hypoxia for 3 h; (iii) hypoxia for 15 h. Nuclei were isolated from cultured C6 cells essentially as described by Dignam et al.(25) . In brief, cells from three 100-mm culture plates were washed twice with ice-cold phosphate-buffered saline, scraped into 6 ml of ice-cold phosphate-buffered saline, and collected by centrifugation at 500 g, for 5 min, at 4 °C. Subsequent steps were performed at 4 °C. The cells were resuspended gently in 1 ml of ice-cold swelling buffer (10 mM Tris-HCl, pH 7.9, 10 mM KCl, 1 mM dithiothreitol), and allowed to stand on ice for 5 min. The swollen cells were centrifuged at 500 g for 5 min, and resuspended in 1 ml of homogenization buffer (10 mM Tris-HCl, pH 7.9, 0.3 M sucrose, 1.5 mM MgCl(2), 0.3% Triton X-100, 1 mM dithiothreitol). After lysis by 10 strokes with a glass Dounce homogenizer, the homogenate was checked under the microscope for cell lysis, and centrifuged at 500 g for 5 min. Nuclei were washed once with homogenization buffer, resuspended in glycerol storage buffer (50 mM Tris-HCl, pH 8.3, 5 mM MgCl(2), 0.1 mM EDTA, 40% glycerol), and frozen in liquid N(2).

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 10^6 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(2), 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 [alpha-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(2), 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 alpha-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 beta-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 10^6 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 beta-actin mRNA. After measurement, the filters were exposed to Kodak XAR-5 film with intensifying screens at -80 °C.

mRNA Stability Assay

The half-life of VEGF mRNA was determined by treating C6 cells with actinomycin D as described by Lindholm et al.(27) . C6 cells were cultured under either normoxia, hypoxia for 3 h, or hypoxia for 15 h, and then actinomycin D was added into the growth medium (10 µg/ml) to block transcription. Immediately after addition of actinomycin D, the cells were returned to the same culture conditions (normoxia or hypoxia). During the following 7 h, cells were harvested every hour, and total RNA was prepared and Northern analysis was performed as described. The amounts of VEGF mRNA and beta-actin mRNA at each time point was quantified after Northern analysis with a PhosphorImager. The amount of VEGF mRNA was corrected for loading differences by the amount of beta-actin mRNA. This correction was confirmed by staining the gel with ethidium bromide (data not shown).

Plasmid Constructs and Transient Transfection

A genomic DNA clone from the human VEGF gene(28) , containing approximately 2.5 kb of 5`-flanking region with the putative promoter and 1 kb of 5`-untranslated region, was provided by Dr. Judith A. Abraham. A 3.2-kb KpnI-NarI fragment, spanning a region from 80 bp up to 3.3 kb upstream of the translation start site of the VEGF gene, was subcloned upstream of the luciferase gene in the promoterless luciferase reporter vector pGL2-Basic Vector (Promega). From this construct, a series of deletion derivatives, including deletion of a 0.9-kb NheI-NarI fragment containing the 5`-untranslated region, were generated as shown in Fig. 4. Hypoxia responsiveness of these constructs in C6 cells was tested by transient transfection assays. A plasmid containing the beta-galactosidase gene driven by the mouse hydroxymethylglutaryl-coenzyme A reductase promoter (29) , a promoter which showed minimal hypoxia responsiveness (data not shown), was used as a control plasmid to standardize transfection efficiency.


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 10^5 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 beta-galactosidase activity. Luciferase activity was measured by AutoLumat LB 953 (Berthold) with Luciferase Assay System (Promega), and beta-galactosidase activity was measured by using chlorophenol red-beta-D-galactopyranoside as substrate(30) . For standardization of transfection efficiency, luciferase activity was divided by beta-galactosidase activity (luciferase/beta-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.


RESULTS

Induction of VEGF mRNA Synthesis in C6 Cells by Hypoxia

A time course of hypoxia-induced changes in VEGF mRNA levels in C6 cells was established by Northern analysis. As shown in Fig. 1, levels of VEGF mRNA began to increase after 3 h incubation of 1% O(2), reached steady-state level after around 15 h, and remained elevated until the end of the period examined (24 h). In several independent experiments, we observed approximately 3-4-fold induction of VEGF mRNA after 3 h of hypoxia and 8-10-fold induction after 15 h of hypoxia. The major transcript size was approximately 4 kb. However, additional other species of VEGF mRNA were detected after longer exposure time. In contrast to VEGF mRNA, neither significant increase nor decrease of beta-actin mRNA by hypoxia was observed up to 15 h of hypoxia in several independent experiments. In some experiments, long lasting hypoxia (longer than 15 h) led to a decrease in beta-actin mRNA, which correlated with a decrease in cell viability (Fig. 1).


Figure 1: Hypoxic induction of VEGF mRNA in C6 cells. C6 cells were incubated either under normoxia or under hypoxia (1% O(2)) for the number of hours indicated. Total RNA (20 µg/lane) was analyzed by Northern analysis using probes for VEGF (top panel) and beta-actin (bottom panel).



Induction of VEGF Gene Expression by Hypoxia Is, in Part, Due to Transcriptional Activation

In order to investigate whether hypoxia activates transcription of the VEGF gene in C6 cells, nuclear run-on assays were performed. Nuclei were prepared from C6 cells cultured under normoxia and under hypoxia for 3 and 15 h. To quantify the transcriptional rate, P-labeled RNAs transcribed from these nuclei were hybridized to plasmids containing murine VEGF cDNA, beta-actin cDNA for standardization, or pBluescript vector DNA as a negative control, which were immobilized on the filters. We observed activation of transcription of the VEGF gene after 3 and 15 h of hypoxia, whereas the transcription of the beta-actin gene was almost unchanged (Fig. 2). In vitro transcription of the VEGF gene was not detected when alpha-amanitin, a potent inhibitor of RNA polymerase II, was included in the reaction mixture (data not shown). The signal intensity of each slot was measured with a PhosphorImager, and the degree of induction of transcription of the VEGF gene by hypoxia was determined after standardization to beta-actin gene transcription. Two independent run-on assays showed that the transcriptional rate of the VEGF gene was elevated 3.4 ± 0.5-fold after 3 h of hypoxic incubation and 2.5 ± 0.6-fold after 15 h of hypoxic incubation.


Figure 2: Nuclear run-on analysis of transcription of the VEGF and beta-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 [alpha-P]UTP, P-labeled RNA (4.5 10^6 cpm) was hybridized to VEGF cDNA, beta-actin cDNA, and pBluescript plasmid immobilized on a nylon membrane.



VEGF mRNA Stability Is Differentially Regulated from Transcriptional Activation

To evaluate the effect of hypoxia on VEGF mRNA stability, we measured half-lives of VEGF mRNA in C6 cells cultured under normoxia and under hypoxia for 3 and 15 h. Cells were treated with actinomycin D in order to block transcription, and the amount of VEGF mRNA in cells thereafter was measured by Northern analysis with a PhosphorImager, and corrected for loading differences by the amount of beta-actin mRNA (Fig. 3). Half-life values for VEGF mRNA obtained from two independent experiments were as follows: under normoxia, 0.89 ± 0.05 h; after 3 h hypoxic treatment, 1.1 ± 0.2 h; after 15 h hypoxic treatment, 2.3 ± 0.2 h. Interestingly, we could not detect significant differences between the half-lives of VEGF mRNA in C6 cells cultured under normoxia and under hypoxia for 3 h. However, the half-life of VEGF mRNA was about 2.6-fold greater in C6 cells after 15 h of hypoxic treatment. These data and the nuclear run-on studies indicate that the initial induction of VEGF mRNA levels in C6 cells after 3 h of hypoxic treatment (3-4-fold, see Fig. 1) is primarily due to transcriptional activation, whereas the late induction after 15 h of hypoxic treatment (approximately 8-10-fold) is due to both transcriptional activation and mRNA stabilization.


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 beta-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 beta-actin mRNA.



Sequences from the 5`-Flanking Region of the VEGF Gene Confer Hypoxic Responsiveness

Based on the results obtained from nuclear run-on assays, we expected cis-acting elements to control the transcriptional activation of the VEGF gene in C6 cells in response to hypoxia. Therefore, we tested several reporter constructs containing 5`-flanking and 5`-untranslated sequences from the human VEGF gene (28) (Fig. 4) for hypoxic responsiveness in a transient transfection assay. C6 cells were transfected with these reporter constructs, and the induction of luciferase activity by hypoxia was calculated as described under ``Experimental Procedures.'' A 131-bp fragment from an ApaI site (-1169; numbered from the translation start site) to the transcription start site(-1038) contained the minimal DNA sequence required for the basal transcription of the human VEGF gene. A 1.2-kb SacI-NheI fragment (-2218 to -984) was shown to be hypoxia responsive, but further deletion of the region between the SacI and BanI sites (-2218 to -1926) deprived the fragment of its hypoxic responsiveness. Thus these studies revealed that a hypoxia responsive element is located in the 293-bp SacI-BanI fragment (-2218 to -1926), and that the 0.9-kb NheI-NarI (-984 to -81) fragment of 5`-untranslated region contains no elements involved in hypoxic induction. These results confirm our result from nuclear run-on assays which showed that hypoxia induces transcriptional activation of the VEGF gene.


DISCUSSION

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(2) treatment, or the hypoxia-induced expression was suppressed in the presence of carbon monoxide(14, 32) . (^3)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.


FOOTNOTES

*
This work was supported in part by the German-Israeli Foundation and the Alexander von Humboldt Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Victoria 3050, Australia.

To whom correspondence should be addressed: Max-Planck-Institut für physiologische und klinische, Forschung, Abteilung Molekulare Zellbiologie, Parkstrabetae 1, 61231 Bad Nauheim, Germany. Fax: 49-6032-72259; Tel.: 49-6032-705278.

(^1)
The abbreviations used are: VEGF, vascular endothelial growth factor; EPO, erythropoietin; Pipes, 1,4-piperazinediethanesulfonic acid; kb, kilobase(s); bp, base pair(s).

(^2)
Breier, G., Clauss, M., and Risau, W., Dev. Dynamics, in press.

(^3)
E. Ikeda and W. Risau, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Dr. Judith A. Abraham (Scios Nova, Inc.) for the generous gift of a genomic clone for human VEGF, and Dr. Dietmar von der Ahe (Max-Planck-Institut, Bad Nauheim, Germany) for technical advice. We also thank Dr. Karl Plate (Klinikum der Albert-Ludwigs-Universität, Freiburg, Germany) and Dr. Eli Keshet (The Hebrew University-Hadassah Medical School, Israel) for valuable discussion.


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