(Received for publication, October 20, 1995)
From the
We describe the genomic organization and functional
characterization of the mouse gene encoding vascular endothelial growth
factor (VEGF), a polypeptide implicated in embryonic vascular
development and postnatal angiogenesis. The coding region for mouse
VEGF is interrupted by seven introns and encompasses approximately 14
kilobases. Organization of exons suggests that, similar to the human
VEGF gene, alternative splicing generates the 120-, 164-, and 188-amino
acid isoforms, but does not predict a fourth VEGF isoform corresponding
to human VEGF. Approximately 1.2 kilobases of 5`-flanking
region have been sequenced, and primer extension analysis identified a
single major transcription initiation site, notably lacking TATA or
CCAT consensus sequences. The 5`-flanking region is sufficient to
promote a 7-fold induction of basal transcription. The genomic region
encoding the 3`-untranslated region was determined by Northern and
nuclease mapping analysis. Investigation of mRNA sequences responsible
for the rapid turnover of VEGF mRNA (mRNA half-life, <1 h) (Shima,
D. T., Deutsch, U., and D'Amore, P. A.(1995) FEBS Lett. 370, 203-208) revealed that the 3`-untranslated region was
sufficient to trigger the rapid turnover of a normally long-lived
reporter mRNA in vitro. These data and reagents will allow the
molecular and genetic analysis of mechanisms that control the
developmental and pathological expression of VEGF.
The mediators of neovascularization comprise a diverse
collection of growth stimulators and inhibitors that have been so
designated because of their abilities to affect angiogenesis in
vivo and/or endothelial cell proliferation in vitro (for
review, see (1) ). Vascular endothelial growth factor (VEGF) ()was initially identified based on its ability to stimulate
vascular permeability (called VPF, for vascular permeability factor)
and was subsequently demonstrated to be an endothelial cell-specific
mitogen and angiogenic factor(2, 3) . In
vivo, VEGF expression has been correlated with embryonic,
physiological, and pathological blood vessel
growth(4, 5, 6) . VEGF's role as a
mediator of angiogenesis has been confirmed in two distinct
pathologies; VEGF has been demonstrated to be a necessary component of
experimental tumor angiogenesis and tumor growth in
rodents(7, 8) , and, more recently, it has been shown
to be causative in the development of ocular angiogenesis secondary to
retinal ischemia(9, 10) .
The spatial and temporal expression patterns of VEGF and its tyrosine kinase receptors, flt-1 and flk-1/KDR, during periods of blood vessel growth have also led investigators to suggest a paracrine role for VEGF during the development of the embryonic vasculature(11, 12) . The VEGF receptor flk-1 is expressed in regions of the early mesoderm, which are presumed to give rise to angioblasts, and is currently the earliest known molecular marker for the endothelial cell lineage. During later stages of embryogenesis, flt-1 and flk-1 receptor mRNA are restricted to the endothelium of vascular cords and blood islands, with VEGF mRNA expressed in adjacent embryonic tissues(13) . Proof of a role for VEGF in vessel development comes from recent studies in which VEGF receptors were deleted by targeted disruption. Mouse embryos, in which the flk-1 receptor was deleted by targeted disruption, lacked blood islands and died between days 8.5 and 9.5. In these embryos, no organized blood vessels were observed and hematopoiesis was dramatically reduced(14) . Mice, in which flt-1 was mutated by targeted disruption, were able to form endothelial cells but unable to assemble them into normal vascular channels and thus died at mid-somite stages(15) .
From these
and other observations, VEGF emerges as a mediator of vasculogenic and
angiogenic events associated with a wide range of biological
events(16) . Consistent with this concept, the local and
systemic signals responsible for orchestrating the growth and
regression of new blood vessels must ultimately regulate VEGF gene
expression. Numerous effectors of VEGF gene expression have been
identified, including cAMP, steroid hormones, protein kinase C
agonists, polypeptide growth factors, oxygen, free radicals, glucose,
cobalt, and iron. The potential mechanisms through which these agents
modulate gene expression are varied, and include transcriptional
regulation through AP-1, AP-2, steroid hormone receptors, p53, and
NFkB, as well as post-transcriptional control of mRNA stability (17, 18, 19, 20, 21) . ()
To begin an analysis of the relevant mechanisms controlling the developmental and pathological expression of VEGF and to develop reagents for defining the role of VEGF in embryonic development using mouse molecular genetics, we have isolated and characterized the mouse VEGF gene. The structure of the gene was determined by restriction mapping, sequencing of intron-exon junctions, definition of the transcription initiation and termination sites, and analysis of the sequence representing the VEGF proximal promoter. Using these structural data, we have assayed the mouse VEGF gene for cis-regions responsible for different aspects of gene regulation and to this end describe gene segments sufficient to promote basal transcriptional activity and post-transcriptional regulation of the VEGF gene.
To isolate
additional clones encompassing the 5`-end of VEGF, a 280-bp cDNA probe
template spanning exons 1-3 was generated, using the polymerase
chain reaction, and used to rescreen 5 10
colonies.
No additional VEGF clones were identified. As an alternative, the exon
1-3 probe was used to screen a 129 mouse genomic library in the
lambda vector EMBL3 (kindly provided by Dr. Richard Moss, Brigham and
Women's Hospital, Boston). The library was screened on charged
nylon membranes (GeneScreen Plus, DuPont NEN) by hybridization at 42
°C in 5
SSPE, 50% deionized formamide, 5
Denhardt's, 10% dextran sulfate, 0.5% SDS, and 100 µg/ml
sheared, denatured salmon sperm DNA. Two positive clones were
identified from screening 1
10
plaques. After two
rounds of plaque purification, phage DNA was isolated and analyzed
further. Restriction digestion and Southern blot analysis of genomic
clones indicated that they encompassed the 5`-end of the VEGF gene; one
clone, designated lambda 8, overlapped with cos15, a cosmid clone that
terminates in the intron upstream of the exon 4 sequence. Sequence
data, restriction maps, and Southern blot analysis of the mouse VEGF
gene were compiled from 9- and 7.5-kb EcoRI subclones that
encompass the VEGF coding region (see Fig. 1).
Figure 1: Genomic organization of mouse VEGF. Restriction map of the mouse VEGF gene and flanking regions as established from the lambda 8 and cosmid 15 clones. Sites are marked for the enzymes AccI (A), BamHI (B), EcoRI (E), HindIII (H), and SmaI (S). Locations of exons 1-8 and intron sizes are indicated. The VEGF open reading frame is indicated by black shading.
To identify the
3`-end of VEGF transcripts, a series of adjacent P-labeled
genomic DNA probes (probe A, 1.3-kb EcoRI fragment; probe B,
750-bp EcoRI-SfiI fragment; probe C, 600-bp SfiI fragment; see Fig. 7), spanning the 3`-end of the
VEGF genomic clone, were hybridized to immobilized mouse lung RNA,
according to standard protocols. Because of a weak, yet significant,
hybridization signal, probe C was predicted to overlap regions of
genomic DNA encoding the VEGF transcript and the 3`-flanking sequence.
A nuclease protection assay was used to more precisely define the
3`-end. A 4.4-kb SmaI genomic DNA fragment (spanning the
3`-UTR and flanking region of VEGF) cloned in pBSII (Stratagene) was
used for riboprobe synthesis. The DNA template was linearized at an NcoI site within the probe B region, and an antisense RNA
probe of 2.2 kb (see Fig. 7) was transcribed with T7 RNA
polymerase according to standard protocols(27) . The probe was
hybridized to C127I mouse mammary epithelial cell total RNA or yeast
tRNA overnight at 30 °C in 40 mM PIPES, pH 6.4, 400 mM NaCl, 80% deionized formamide, 1 mM EDTA. The mixture was
diluted in nuclease digestion buffer (50 mM sodium acetate, pH
5.0, 30 mM NaCl, 1 mM zinc acetate, 20 µg/ml
denatured calf thymus DNA) and then incubated in the presence of 300
units of mung bean nuclease (Life Technologies, Inc.) for 1 h at 30
°C. Reaction products were separated by denaturing gel
electrophoresis in an 8 M urea, 4% polyacrylamide gel and
visualized by autoradiography.
Figure 7: Analysis of the mouse VEGF 3`-end. A, schematic of exon 8 and corresponding flanking region. EcoRI (E), SmaI (S), and NcoI (Nc) restriction sites are marked. Regions corresponding to 3`-UTR probes A, B, and C and the T7-synthesized riboprobe are indicated. B, slot-blot hybridization analysis of 10 or 2 µg of mouse total RNA (m) and control yeast tRNA (y) with 3`-UTR probes. C, analysis of mung bean nuclease-digested C127I RNA/riboprobe hybrids. A 510-520-nucleotide digestion-resistant product is present solely in total RNA from hypoxic C127I cultures.
Figure 6: Functional analysis of the mouse VEGF promoter. Relative luciferase activities were obtained from transiently transfected VEGF promoter-luciferase constructs in C6 glioma cells. Schematic representation of assay constructs (see ``Materials and Methods'') is shown. All luciferase values were normalized for transfection efficiency, using placental alkaline phosphatase activity, and are expressed as the level of luciferase activity relative to the activity of the promoterless luciferase control plasmid pGL2 basic. Data shown are from duplicate analyses and are representative of five separate experiments. Within these five experiments, peak VEGF promoter activity varied from 7 to 10-fold relative to negative controls.
Actinomycin D chase assays and Northern blot
analysis of total RNA were performed as described(20) .
Briefly, confluent cells were incubated in culture media containing
actinomycin D (5 µg/ml) and subsequently incubated for 0-8 h
in standard culture conditions. Total RNA was extracted by the modified
acid-phenol method using RNAzol B (Tel-test) and analyzed by Northern
blot for LTR-neo, LTR-VEGF, and -actin mRNA levels. 15 µg of
RNA/sample were fractionated by denaturing gel electrophoresis and
capillary blotted to charged nylon (GeneScreen Plus). Prehybridization
and hybridization were carried out in 6
SSPE, 5
Denhardt's, 50% formamide, 1% SDS, and 100 µg/ml sheared,
denatured salmon sperm DNA. Probes for Northern blot analysis were
random prime labeled with
P, using the following
templates: a 280-bp fragment encoding exons 1-3 of the mouse VEGF
cDNA open reading frame, a 440-bp BssHII-SmaI
fragment of the neo gene, and a 400-bp fragment from the
3`-UTR of human
-actin(30) .
A restriction map for the two overlapping clones was assembled by single, double, and partial digestions with EcoRI, BamHI, AccI, HindIII, and SmaI restriction enzymes (Fig. 1). The locations of exons relative to the restriction map were established by nucleotide sequencing the restriction sites proximal to exons and Southern blot analysis of cloned DNA with exon-specific probes (data not shown). Restriction enzyme analysis of genomic DNA by Southern blot was used to confirm mapping data and verified that mouse VEGF was encoded as a single copy gene (Fig. 2). The overlapping genomic clones define a contiguous stretch of 45 kb of DNA, of which approximately 14 kb represents the mouse VEGF coding region.
Figure 2: Genomic Southern blot analysis of mouse VEGF. 129 mouse genomic DNA (10 µg) was digested with the indicated enzymes and analyzed in a Southern blot using a mouse VEGF genomic DNA probe (probe A as illustrated in Fig. 1). The hybridizing fragment in HindIII-digested DNA is less than 3.0 kb in size and has been run from the gel.
Figure 3: Nucleotide sequence for mouse VEGF intron-exon borders and the sequence surrounding the mouse VEGF transcription initiation site. Intron and UTR sequence is shown in lower case letters, coding sequence is shown in upper case letters, and the VEGF translation start and stop codons are boxed. An arrow indicates the initiation site of RNA synthesis and is designated +1. Consensus binding sites for relevant transcription factors are marked as follows: AP-1, thin line; AP-2, heavy hatched line; NFkB, broken line; Sp1, heavy line.
A fourth human VEGF isoform, designated
VEGF, was previously identified by polymerase chain
reaction amplification of VEGF isoforms from a fetal human liver cDNA
library(31) . From a comparison of cDNA and genomic sequence,
this splice variant was predicted to be derived from the utilization of
an alternative splice donor site downstream from the site originally
identified for exon 6. The mouse gene would not be predicted to encode
an isoform homologous to human VEGF
. When compared to the
published human sequence, the sequence of the corresponding region of
the mouse gene contains an additional nucleotide, creating a frameshift
that results in an in-frame stop codon (Fig. 4).
Figure 4:
Comparison of mouse exon-intron 6 with the
corresponding region of human VEGF. The nucleotide insertion in the
human sequence that creates a continuous open reading frame in
VEGF is marked. The translation stop codon present in the
homologous region of mouse VEGF genomic DNA is designated with an asterisk. Predicted amino acids are indicated below the
nucleotide sequence using a single letter
format.
Figure 5:
Primer extension analysis of the VEGF
transcription start site. A, Northern blot analysis of total
RNA from normoxic and hypoxic cultures of C127I demonstrating the
differences in steady-state VEGF mRNA levels. B, primer
extension products were separated by electrophoresis on a denaturing
polyacrylamide gel along with a DNA sequencing ladder generated with
the same oligonucleotide used for primer extension. Poly(A) and poly(A)
RNA from normoxic (N) or
hypoxic (H) cultures of C1271 mouse mammary cells were used in
the reactions. A single primer extension product was detected in the
poly(A)
RNA, indicating the location of the
transcriptional start site (the complementary nucleotide is shown by an asterisk).
To determine if the sequences upstream of the transcription initiation site are sufficient to direct transcription, a 1.6-kb fragment, including 1.2 kb of 5`-flanking region and 0.4 kb of 5`-UTR, was fused in both orientations to a promoterless luciferase transcription reporter gene and examined for the ability to mediate basal transcription. In addition, 5`-deletions in the putative promoter region were also monitored for their effect on reporter activity.
The murine astrocytoma cell line, C6, was transiently transfected with reporter constructs, and cell extracts were assayed for luciferase activity 48 h post-transfection. VEGF sequences fused to the reporter in the appropriate transcriptional orientation consistently produced a 7-fold increase in luciferase activity when compared to a promoterless luciferase construct (Fig. 6). In contrast, VEGF sequences fused in the opposite transcriptional orientation did not induce a significant level of reporter activity. Deletion of 445 or 770 bp from the 5`-end of the promoter fragment resulted in a 25% decrease in reporter activity, whereas a 1.3-kb deletion, which removed putative promoter sequences and the transcriptional initiation site, reduced luciferase activity to background levels.
Nuclease protection analysis of mRNA from C127I cells was used to obtain more precise information on the 3`-end of VEGF transcripts. A 2.2-kb radiolabeled antisense riboprobe was generated from a genomic DNA template and hybridized to total RNA from hypoxic and normoxic cultures of C127I and a yeast tRNA negative control. Following nuclease digestion and electrophoretic separation of nuclease products, a 510-520-nucleotide nuclease-resistant species was observed in hypoxic RNA hybrids (Fig. 7C). The same protected fragment was observed from normoxic RNA hybrids after extended exposures of the analytical gel, reflecting the 10-20-fold differences in VEGF mRNA levels between normoxic and hypoxic cultures. The faint band of about 450 bp may reflect the existence of a less utilized alternative termination site. No product was seen in the yeast tRNA negative control. These data place the site of transcriptional termination approximately 510 bp downstream from the riboprobe terminus, leading to a 3`-UTR of approximately 2.2 kb.
To investigate if a determinant of mRNA destablization is present in the VEGF 3`-UTR, genomic sequences corresponding to this region, including the putative polyadenylation signal, were fused to a neomycin (neo) reporter mRNA (designated LTR-VEGF). The VEGF 3`-UTR replaces SV40 DNA sequences that normally terminate the neo transcripts. The neo/SV40 mRNA fusion (designated LTR-neo) is normally quite stable, with a half-life of >8 h. The addition of destabilization sequences from the 3` UTR of granulocyte-macrophage colony stimulating factor, c-fos, and c-myc to LTR-neo mRNA has been shown to direct its rapid decay(29) .
Actinomycin D chase
studies were used to compare the rate of decay for LTR-VEGF mRNA to
that of LTR-neo mRNA (control) in the C127I mammary epithelial
cell line. As expected, the control LTR-neo mRNA remained
stable throughout an 8-h period in the absence of transcription (Fig. 8). In contrast, LTR-VEGF fusion mRNA behaved similar to
the endogenous VEGF mRNA, with both VEGF and LTR-VEGF transfectants
undergoing rapid decay with half-lives of less than 1 h. Levels of
endogenous -actin mRNA, a transcript with an average half-life,
were relatively stable under each experimental condition over the time
course of the experiment(33) .
Figure 8:
The VEGF 3`-UTR contains a region that
promotes destablization of a normally stable fusion mRNA. LTR-neo and LTR-VEGF transfectants (schematic of fusion mRNA constructs
shown; see ``Materials and Methods'') were used to analyze neo fusion mRNA, VEGF mRNA, and -actin mRNA decay using
an actinomycin D chase protocol. Time (h) after the addition of
actinomycin D is marked above each lane.
VEGF has been implicated as a multi-functional effector of vascular cell function. In addition to its well documented angiogenic properties, VEGF is also a potent stimulator of leukocyte migration, vascular permeability, and procoagulant functions in endothelium(34, 35) . Moreover, the presence of significant VEGF mRNA and protein in various tissues of the adult suggests an additional role for VEGF in the maintenance of normal vascular cell integrity and/or behavior(4) . How VEGF gene expression is regulated during the transition from periods of vascular quiescence to active vascular growth, remodeling, and repair is not understood. To begin to investigate the structure-function relationships critical for the regulation of VEGF expression, we have characterized the mouse VEGF gene.
Since
little is known about the physiological roles of the four VEGF
isoforms, it is difficult to predict the functional significance of a
divergence in isoform generation between humans and mice. The putative
VEGF isoform was identified using the polymerase chain
reaction to amplify VEGF-related cDNAs and consists of an alternative
splice variant with an extended exon 6 region that results in a
17-amino acid insertion relative to the VEGF
isoform.
Analysis of VEGF synthesis, secretion, and bioactivity in vitro has revealed that an engineered VEGF
protein shares
similar biochemical and functional properties with VEGF
,
suggesting that these two isoforms could provide redundant biological
functions(31) . To date, the biochemical and biological
descriptions of VEGF
have been sparse and have relied on
data obtained from the fusion of the N-terminal region of VEGF
to the partial VEGF
cDNA clone; the expression of
native VEGF
mRNA or protein by tissue culture cells or in vivo has not been adequately described.
Transient transfection assays indicate that a 1.2-kb segment of 5`-flanking region specifically directs the transcription of a reporter gene in VEGF-producing cells. Deletion of the 1.2-kb region, including the putative transcription initiation site, abolished promoter activity. Results from these functional analyses support transcript mapping and sequence analysis data that identify this DNA segment as the VEGF proximal promoter.
Transfection of C6 rat glioma with constructs deleting either 445 or 770 bp from the 5`-end of the 1.2-kb promoter region resulted in a similar 25% decrease in reporter activity, suggesting that cis-acting elements necessary for basal promoter activity in C6 cells reside in the 450-bp DNA segment deleted from the 5`-end of the promoter fragment. Yet, a relevant promoter activity resides within the first 450 bp upstream to the VEGF gene. Further studies will be required to identify and characterize the cis- and trans-acting components necessary for both basal and inducible regulation of VEGF gene transcription.
Sequence analysis of the 1.2-kb region, upstream of the transcription initiation site, revealed the presence of a number of potential cis-acting regulatory elements. Similar to the human VEGF gene, multiple consensus binding sites for AP-1 and AP-2 transactivating complexes are present. AP-1 activity has been shown to be stimulated by phorbol esters and growth factors, and both cAMP-dependent kinase and protein kinase C pathways have been implicated in the activation of AP-2(40, 41) . Phorbol esters, peptide growth factors, and intracellular elevation of cAMP also induce steady-state VEGF mRNA, suggesting that the AP-1 and AP-2 consensus sites present in the VEGF promoter may mediate VEGF transcriptional activation in response to these effectors(17, 21) . In addition, studies from a number of laboratories indicate that in some cells transcriptional activation plays a role in the up-regulation of VEGF mRNA by hypoxia(42, 43, 44) . Further, the site of transcriptional initiation and numerous regions within the proximal promoter of mouse VEGF share significant similarity in both sequence and organization with the human homologue. The conserved organization of transcriptional regulatory sequences within the two promoters may imply a critical role for these regions in the proper regulation of VEGF gene expression. In contrast, consensus sites for NFkB, a transactivator implicated in the regulation of inflammation and stress response genes(45) , are located 90 and 185 bp upstream of the initiation site in mouse VEGF, whereas NFkB consensus sites have not been identified in the human VEGF promoter.
Experimental conditions that induce VEGF mRNA, such as phorbol ester treatment or hypoxia, are known to regulate mRNA stability (20, 32) . Investigation of the cellular mechanisms controlling transcript stability suggest that certain mRNAs contain distinct structural elements for positive and negative regulation of mRNA stability. Specific destablization sequences vary considerably but usually consist of AU-rich elements in the 3`-UTR of unstable mRNAs (46) . Multiple AU-rich regions exist throughout the 3`-UTR of mouse VEGF (data not shown) and represent potential candidates for destabilizing sequences. Less is known about sequences that selectively or inducibly promote mRNA stability. For the transferrin receptor, a well studied model of inducible mRNA stability, the 3`-UTR contains a stem-loop sequence that interacts with inducible cellular factors to promote mRNA stability(47) . During periods of iron starvation, RNA-protein interactions at the stem-loop sequence are dominant over the effects of a distinct region of AU-containing sequences, which otherwise trigger transferrin receptor mRNA degradation(48) . Further investigation will be required to define sequences within the VEGF mRNA required for positive and negative regulation of post-transcriptional mRNA stability.
The findings reported here provide a framework for the comprehensive analysis of the regulation of VEGF expression and VEGF structure-function relationships. Such studies will be critical to understanding and eventually modulating the role of VEGF during physiological and pathological blood vessel growth.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U41383[GenBank].