©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Rat Vascular Endothelial Growth Factor Gene by Hypoxia (*)

Andrew P. Levy (1), Nina S. Levy (2), Scott Wegner (2), Mark A. Goldberg (2)(§)

From the (1) Cardiology Division and (2) Hematology-Oncology Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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,() 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) .

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.


MATERIALS AND METHODS

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).

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 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.


RESULTS

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.

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. 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.




DISCUSSION

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) () 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.

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 3.7 kb in length, in good agreement with Northern blot results.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants T32HL07604 (to A. P. L.), 1F32HL08838-02 (to N. S. L.), and DK45098 (to M. A. G.), an American Heart Association Established Investigator award (to M. A. G.), and an American Heart Association grant-in-aid (to M. A. G.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U22372 and U22373.

§
To whom correspondence and reprint requests should be addressed: Brigham and Women's Hospital, LMRC Rm. 222, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-7646; Fax: 617-739-0748.

The abbreviation used are: VEGF, vascular endothelial growth factor; Epo, erythropoietin; HIF-1, hypoxia-inducible factor 1; SSC, sodium saline citrate; SV40, simian virus 40; TBE, Tris borate EDTA; AP-1, activator protein 1; AP-2, activator protein 2; bp, base pair(s); kb, kilobase(s); UTR, untranslated region; EMSA, electromobility shift assay.

A. P. Levy, N. S. Levy, and M. A. Goldberg, unpublished observations.


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