(Received for publication, September 5, 1995; and in revised form, November 16, 1995)
From the
The major control point for the hypoxic induction of the vascular endothelial growth factor (VEGF) gene is the regulation of the steady-state level of the mRNA. We previously demonstrated a discrepancy between the transcription rate and the steady-state mRNA level induced by hypoxia. This led us to examine the post-transcriptional regulation of VEGF expression. Actinomycin D experiments revealed that hypoxia increased VEGF mRNA half-life from 43 ± 6 min to 106 ± 9 min. Using an in vitro mRNA degradation assay, the half-life of VEGF mRNA 3`-untranslated region (UTR) transcripts were also found to be increased when incubated with hypoxic versus normoxic extracts. Both cis-regulatory elements involved in VEGF mRNA degradation under normoxic conditions and in increased stabilization under hypoxic conditions were mapped using this degradation assay. A hypoxia-induced protein(s) was found that bound to the sequences in the VEGF 3`-UTR which mediated increased stability in the degradation assay. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the hypoxia-induced stabilization of VEGF 3`-UTR transcripts and inhibited hypoxia-induced protein binding to the VEGF 3`-UTR. These findings demonstrate a significant post-transcriptional component to the regulation of VEGF.
Hypoxia has been shown to be an important stimulus for the new
blood vessel formation seen in coronary artery disease(1) ,
tumor angiogenesis(2) , and diabetic
neovascularization(3) . VEGF, ()also known as
vascular permeability factor, is a potent angiogenic and endothelial
cell-specific mitogen (4, 5, 6) , which is
regulated by hypoxia in vitro(2, 7, 8) and in
vivo(2, 3, 9, 10, 11) .
The major control point for the hypoxic induction of the VEGF gene is
the regulation of the steady-state level of mRNA (2, 8) which is determined by the relative rates of
mRNA synthesis and decay.
We have previously demonstrated that hypoxia induces VEGF steady-state mRNA 25.0 ± 11.4 and 12.0 ± 0.6 fold in rat primary cardiac myocytes (8) and rat pheochromocytoma PC12 cells(12) , respectively. However, nuclear runoff transcription assays demonstrated that the transcription rate for VEGF was increased only 3.1 ± 0.6-fold by hypoxia in the PC12 cells(12) . Rat genomic sequences encoding VEGF were cloned and a 28-bp element in the 5` promoter was identified that mediates a significant portion of this hypoxia-inducible transcription in transient expression assays. This element was shown to have sequence and protein binding similarities to the hypoxia-inducible factor 1 binding site within the erythropoietin (Epo) 3` enhancer(12) . These studies demonstrated that, while increased transcription rate can account for a portion of the increase in the steady-state level of VEGF mRNA in the PC12 cells, it does not account for all of the increase and suggested that a post-transcriptional mechanism plays a significant role in the hypoxic induction of VEGF mRNA, as well.
Post-transcriptional mechanisms of regulation have previously been suggested for Epo (13, 14, 15) and demonstrated for tyrosine hydroxylase (16) , two other hypoxia-inducible genes. In the present study we examine the post-transcriptional regulation of VEGF mRNA expression under both normoxic and hypoxic conditions. We have employed several complementary techniques including actinomycin D chase experiments, in vitro mRNA degradation studies, and RNA electromobility shift assays.
[P]CTP-labeled,
capped, and polyadenylated transcripts were synthesized in
vitro(18) . The EcoRI site of pSP64 poly(A)
(Promega) was transformed into an AseI restriction enzyme site
by filling in EcoRI-digested pSP64 poly(A) with the Klenow
fragment and then blunt-end ligating the vector. Restriction fragments
containing the 3`-UTR of VEGF derived from clone 11.4 (12) were
cloned into the multiple cloning site of this modified pSP64 vector. A
series of deletions were made from the 3` end of these sequences using
unique restriction sites in the VEGF 3`-UTR. Digestion of these
plasmids with AseI-generated DNA templates containing a
poly(dT) sequence that was transcribed into a 30-base long poly(A) tail
at the 3` end. Capped transcripts were synthesized from these templates
with SP6 RNA polymerase using the MEGAscript in vitro transcription kit (Ambion, Austin, TX) according to the
manufacturer's protocol with a 4:1 ratio of m
G5`pppG
(cap analog) to GTP. Labeled RNA transcripts were produced by inclusion
of [
-
P]CTP (3000 Ci/mmol) in the reaction.
80,000 cpm were used for each degradation assay.
Degradation assays
were performed by incubating the transcript (10 cpm) with
130 µg of cytoplasmic extract in a total volume of 39 µl in a
master mix at room temperature. At each time point the reaction was
stopped by transferring 3 µl from this master mix to a tube
containing 15 µl of H
O and 2 µl of 5 M NH
OAc with 100 mM EDTA. The sample was
extracted once with phenol:chloroform:isoamyl alcohol (25:24:1), and
the supernatant was precipitated with 20 µl of isopropanol. The
pellets were washed once with 80% ethanol and air-dried. Samples were
then electrophoresed on a formaldehyde-agarose gel and transferred to
GeneScreen (DuPont NEN). Quantitation of the remaining primary
(undegraded) transcript at the different time points was performed with
a Molecular Dynamics PhosphorImager. All time points were performed in
triplicate.
5 mg of actinomycin D were initially dissolved in 1
ml of MeSO and subsequently diluted with DMEM to a
concentration of 50 µg/ml (10
) stock solution. Flasks were
harvested for RNA at 0, 5, 10, 15, 30, 60, 120, 240, and 480 min after
the addition of actinomycin D. Total RNA was prepared from the flasks
using RNA STAT-60 (Tel-Test ``B,'' Inc., Friendswood, TX) and
isolated according to the manufacturer's protocol.
VEGF and 18
S rRNA were detected by RNase protection analysis of 10 µg of RNA
isolated at the various time points. RNase protection assays were
performed as described previously (8) to specifically detect
the VEGF isoform and 18 S rRNA. After electrophoresis on
6% polyacrylamide, 7 M urea gels, the protected fragments were
quantitated using a PhosphorImager (Molecular Dynamics). The quantity
of VEGF mRNA was normalized to the amount of 18 S rRNA (19) by
calculating a VEGF/18 S ratio for each sample. All time points were
performed in triplicate. The entire experiment was repeated three
separate times. The half-life of VEGF mRNA was calculated by drawing
the best fit linear curve on a log-linear plot of the VEGF/18 S ratio versus time. The time at half-maximal VEGF/18S ratio was taken
to be the half-life.
A series of
overlapping oligonucleotides was used to prepare templates for the
generation of short transcripts containing the putative constitutive
RNA-protein binding site. Competitor WT (VEGF nucleotide
1050-1080, GenBank
accession no. U22372) was
generated by overlapping oligonucleotides T7A
(5`-gcggatccTAATACGACTCACTATAGGGAGGTGTGTGAGTGGCTTACCCTTCCCCATTTTC-3`)
(VEGF nucleotide 1050-1080, GenBank
accession no.
U22372) and B (5`-ccgattcGAAAATGGGGAAGGGTAAGCCACTCACACA-3`)(VEGF
nucleotide 1080-1051, GenBank
accession no. U22372).
Competitor WT
(VEGF nucleotide 1050-1091,
GenBank
accession no. U22372) was generated by overlapping
oligonucleotides T7A and C
(5`-cCCTTGGGAAGGGAAAATGGGGAAGGGTAAGCCACTCACACA-3`) (VEGF nucleotide
1091-1051, GenBank
accession no. U22372). Competitor
M (VEGF nucleotide 1050-1080 containing a 3-bp change in the VEGF
sequence, GenBank
accession no. U22372) was generated by
overlapping oligonucleotides T7A and D (5`-
ccgattcGAAAATGGGGACTTGTAAGCCACTCACACA-3`) (VEGF nucleotide
1080-1051, GenBank
accession no. U22372). Equimolar
amounts of each of the oligonucleotides was annealed with its partner
and then treated with Klenow fragment to fill in the overhang. These
oligonucleotide generated templates then were used to make RNA
transcripts with T7 RNA polymerase according to the
manufacturer's protocol for short transcripts (Ambion).
Radiolabeled RNA transcripts (100,000 cpm/reaction) with or without
nonradioactive competitor were incubated with 20 µg of S100
cytoplasmic extract for 15 min at room temperature. 25 units of
ribonuclease T1 were then added followed 10 min later by heparin to a
final concentration of 5 mg/ml. Electrophoresis of RNA-protein
complexes was carried out on 7% native polyacrylamide gel
(acrylamide/methylene bisacrylamide ratio, 30:1) with 0.5 TBE
(30 mM Tris, 30 mM boric acid, 0.06 mM EDTA,
pH 7.3 at 20 °C) at 4 °C. The gels were preelectrophoresed for
1 h at 35 A followed by electrophoresis of RNA-protein complexes for
1.5 h at 30 A. Gels were dried and exposed to x-ray film (Kodak). The
RNA-protein bands were quantitated by PhosphorImager analysis
(Molecular Dynamics).
Figure 1:
VEGF mRNA levels following treatment
with actinomycin D. The time course, RNA harvest, and analysis was
performed as described under ``Materials and Methods.'' Data
are shown from a representative experiment with each time point
performed in triplicate. VEGF RNA is normalized to 18 S rRNA. The
experiment was performed three times. , hypoxia;
,
normoxia.
Figure 2:
Mapping instability elements in the VEGF
3`-UTR under normoxic conditions.
[P]CTP-labeled, capped, and polyadenylated
transcripts were generated in vitro as described under
``Materials and Methods.'' A, restriction map of
constructs in pSP64A (AseI). Linear fragments (A-D) were cloned in the pSP64A (AseI) vector
and used to generate sense 3`-UTR transcripts in vitro.
Deletions from the 3` end of the UTR were produced with the designated
restriction enzymes. Construct A (full-length) (nucleotide
1-2201, GenBank
accession no. U22372) contains the
entire 3`-UTR and yields a RNA of 2.2 kb. Construct B (XbaI) (nucleotide 1-1754, GenBank
accession no. U22372) is derived by deletion of the XbaI
from construct A and yields a RNA of 1.7 kb. Construct C (NsiI) (nucleotide 1-1255, GenBank
accession no. U22372) is derived by deletion of the NsiI-XbaI fragment from construct B and yields a RNA
of 1.2 kb. Construct D (StuI) (nucleotide
1-913, GenBank
accession no. U22372) is derived by
deletion of the StuI-XbaI fragment from construct B
and yields a RNA of 900 bases. The locations of the nonameric
instability consensus signals are depicted by lines with open circles. The half-life in minutes obtained for each
construct was expressed relative to the half-life of construct A for
each individual experiment. Results are expressed as the mean ±
S.E. of four different experiments. In the experimental conditions
described under ``Materials and Methods'' with S100 extract
from normoxic cells, the half-life of construct A was approximately 3
min. B, representative autoradiograph of products from a
cell-free degradation assay of constructs A (Full-length) and
D (StuI) as described under ``Materials and
Methods.'' Time refers to the time after the addition of normoxic
cytoplasmic extract to the RNA. The arrow points to the
undegraded transcript. C, log-linear regression lines of VEGF
RNA degradation quantitated by PhosphorImager analysis. The half-life
of each construct was calculated from the regression line extrapolated
to time 0. In the representative experiment shown, the half-life of
construct A (full-length,
) was 2.6 min, B (XbaI,
) was 2.0 min, C (NsiI,
) was 4.0 min, and D (StuI,
) was 8.0 min.
Figure 3:
Mapping an element in the VEGF 3`-UTR that
mediates stabilization by hypoxia.
[P]CTP-labeled, capped, and polyadenylated
transcripts were generated in vitro as described under
``Materials and Methods.'' A, restriction map of
constructs A-D in pSP64A (AseI) as described in
the legend to Fig. 2A. A half-life for each construct
was determined with normoxic and hypoxic extracts using the identically
labeled transcript. The results are expressed as a ratio of the
transcript half-life using hypoxic to normoxic extracts. All of the
time points were performed in triplicate. Each transcript was assayed
three different times with different extracts. B,
representative autoradiograph of products from the degradation assay of
constructs A (Full-length) and D (StuI). Time refers
to time after the addition of the normoxic or hypoxic extract to the
labeled transcript. The arrow points to the undegraded
transcript. One of the RNA pellets in the triplicate 5 min 1% O
time point for the StuI RNA fragment was lost in
processing, and the data from this sample are not included. C,
log-linear regression lines of VEGF RNA degradation quantitated by
PhosphorImager analysis. The half-life of each construct was calculated
from these regression lines using normoxic (
) and hypoxic
(
) extracts. This is a representative experiment of the data
summarized from three independent experiments in Fig. 3A. Each time point was performed in
triplicate.
Figure 4:
Identification of constitutive and hypoxia
inducible RNA-protein complexes by EMSA. A, map of the VEGF
mRNA 3`-UTR demonstrating location of templates used for generation of
riboprobes for EMSA and to map the cis-elements with which the RNA
binding proteins interact. A T7 promoter was appended to the sense
primer for generation of templates as described under ``Materials
and Methods.'' The StuI-NsiI template
corresponds to nucleotide 909-1279 of the VEGF 3`-UTR, GenBank accession no. U22372. The NsiI transcription termination (TT) site template includes nucleotide 1251-1877 of the
VEGF 3`-UTR, GenBank
accession no. U22372. Restriction
endonuclease MseI, HinfI, EcoRI, and XbaI sites in the NsiI-TT site template are located
at nucleotides 1412, 1566, 1632, and 1754, respectively, of the VEGF
mRNA 3`-UTR, GenBank
accession no. U22372. TGA is the
translation termination codon of VEGF and is located 6 bp 5` to
nucleotide 1 in GenBank
accession no. U22372. TT is the
transcription termination site of VEGF mRNA. The nonameric instability
consensus signals are depicted by lines with open
circles. The small open box at nucleotide 1070 is the proposed
site to which the constitutive protein complex binds. B, EMSA
of the constitutive RNA-protein complex. RNA EMSA using the NsiI-StuI fragment as template was performed as
described under ``Materials and Methods.'' Unlabeled RNA
transcripts for competition studies were generated from the following
templates: NsiI-StuI (VEGF);
IRE(22) ; tyrosine hydroxylase (TH) 162-bp
fragment(16) ; oligonucleotides WT
, WT
,
and M as described under ``Materials and Methods.''
Proteinase K (PK) indicates extracts were first treated with
proteinase K before adding the probe. The arrow points to the
constitutive RNA-protein complex. The bracket encompasses free
and degraded probe. C, sequence homology. Region of homology
between the rat VEGF 3`-UTR, rat tyrosine hydroxylase 3`-UTR, and human
Epo 3`-UTR. This region of the NsiI-StuI fragment of
the VEGF 3`-UTR (nucleotide 1066-1075) was demonstrated to bind a
protein(s) in S100 cytoplasmic extracts. The tyrosine hydroxylase
sequence is within a 28-bp sequence of the tyrosine hydroxylase 3`-UTR
(nucleotide 1552-1579) (32) that is protected by a
hypoxia-inducible protein(16) . The Epo sequence (nucleotide
2831-2841) (33) is within a 120 bp sequence of the Epo 3`
UTR shown to bind a Epo mRNA binding protein that is up-regulated by
hypoxia in brain and spleen(15) . Nucleotide sequence for rat
VEGF, rat tyrosine hydroxylase, and human Epo refer to GenBank
accession nos. U22372, M10244, and M11319, respectively. D, EMSA of the hypoxia-inducible complex. RNA EMSA using the NsiI-transcription termination fragment generated by PCR or
agarose gel-purified restriction endonuclease digested subfragments of
this fragment as templates for the generation of probe (labeled RNA
transcript). [
P]CTP labeled RNA transcripts were NsiI-transcription termination (N), NsiI-XbaI (X), NsiI-EcoRI (R), NsiI-HinfI (H), or NsiI-MseI (M). Unlabeled competitors were
the NsiI-transcription termination transcript (N) or
IRE transcripts (22) present in 100
molar excess to the
labeled probe. Similar results were obtained with four different
preparations of S100 extract. The arrows point to the
hypoxia-inducible complex. The bracket encompasses the free
and degraded probe.
A
hypoxia-inducible protein complex was mapped by EMSA between the NsiI site and the transcription termination site (Fig. 4A) using a template generated by a strategy
described under ``Materials and Methods.'' The RNA-protein
complex was induced 2.2 ± 0.2-fold (n = 12) by
EMSA using hypoxic versus normoxic S100 extracts (Fig. 4D). This complex could be inhibited by excess
unlabeled transcript from this region, but was not displaced by a
500-fold excess of IRE transcript (Fig. 4D) or Epo
transcripts. Proteinase K treatment of the extracts completely
inhibited formation of the complex. 3` truncated forms of this template
were generated using restriction endonucleases XbaI, EcoRI, HinfI, and MseI (Fig. 4A). RNA transcripts from these truncated
templates allowed the binding site for this hypoxia-inducible species
to be further defined within a MseI-XbaI fragment
(nucleotide 1412-1754, GenBank accession no. U22372) (Fig. 4D).
Figure 5:
Genistein inhibits hypoxic stabilization
of VEGF 3`-UTR transcripts in vitro. Cells were pretreated
with 500 uM genistein, an inhibitor of the hypoxic induction
of VEGF mRNA(32) , for 30 min prior to beginning the hypoxic
exposure. After 4 h extracts were prepared from normoxic and hypoxic
cells. Degradation of full-length VEGF 3`-UTR transcript (construct A, Fig. 2A) was assessed as described under
``Materials and Methods.'' A, representative
autoradiograph of decay kinetics of the VEGF 3`-UTR with genistein
treated or control cells using hypoxic and normoxic extracts. The arrow points to undegraded transcript. One of the RNA pellets
in the triplicate 10 min 1% O time point for genistein
treated cells and one of the RNA pellets in the triplicate 10 min 21%
O
time point for control cells were lost in processing and
the data from these samples are not included. B, regression
analysis of A demonstrating an inhibition in the stabilization
of VEGF 3`-UTR transcripts in extracts prepared from genistein-treated
cells. The experiment was performed three times with different
preparations of extract. In the representative experiment shown, the
ratio of the half-lives in hypoxia (
) to normoxia (
) of
the VEGF 3`-UTR transcript is decreased from 1.4 in control cells to
0.5 in genistein-treated cells. C, genistein inhibited
formation of the hypoxia-inducible RNA-protein complex on EMSA. The NsiI-transcription termination template was used for EMSA
analysis as described under ``Materials and Methods.'' The
competitor was an RNA transcript derived from the NsiI-transcription termination template (VEGF) or the
IRE element(22) . 1 G or 21 G indicates that
the S100 extract was made under hypoxic or normoxic conditions,
respectively, from cells treated with genistein. The arrow points to the hypoxia-inducible
species.
Hypoxia exerts its control on VEGF gene expression by increasing VEGF steady-state mRNA levels. Previous work has strongly suggested that an increase in transcription rate of the VEGF gene cannot account for all of the observed increase in the steady-state VEGF mRNA levels induced by hypoxia(12) . These studies provide further evidence for a post-transcriptional mechanism contributing to VEGF mRNA induction by hypoxia. Several cis-acting elements that may mediate the turnover of VEGF mRNA under normoxic and hypoxic conditions are identified.
The half-life of the VEGF mRNA, determined using actinomycin D, is increased 2.5 ± 0.4-fold by hypoxia. Steady-state kinetics (27) would predict that the increase in steady-state mRNA with hypoxia would be the product of the increase in the transcription rate and the increase in the mRNA half-life. These data therefore provide an adequate explanation for the discrepancy between the increase in the steady-state mRNA (12.0 ± 0.6) and the increase in the transcription rate (3.1 ± 0.6) in PC12 cells.
We have demonstrated using an in vitro RNA degradation assay that there are two distinct cis-acting instability elements in the VEGF 3`-UTR. The VEGF 3`-UTR contains two consensus nonameric sequences 5`-UUAUUUA(U/A)(U/A)-3` (25, 26) that have been demonstrated to mediate the rapid turnover of multiple cytokine mRNAs. These nonameric consensus sequences fall within the fragments shown to significantly affect transcript stability in the in vitro degradation assays. We cannot rule out however that other sequences contained within these fragments are responsible for or contribute to the RNA instability.
The increased stability of VEGF 3`-UTR transcripts in vitro in the presence of hypoxic versus normoxic extracts has allowed us to map an element that when deleted from the UTR abrogates this increased stability with hypoxic extracts. From these studies one may hypothesize that a trans-acting factor that mediates stability binds to this region or, alternatively, the region is necessary for formation of a RNA secondary structure that mediates the change in RNA stability.
In EMSA studies, the region of the VEGF mRNA 3`-UTR to which the hypoxia-induced protein complex bound correlated with the RNA degradation assays and points toward an important role for this complex in mediating the increased stabilization of VEGF mRNA by hypoxia. The hypoxia-inducible complex is occasionally seen to migrate as a doublet using the entire NsiI transcription termination site riboprobe (Fig. 4D). This has not been observed with truncated forms of the template (NsiI-EcoRI), although hypoxia-inducible binding is still seen. In addition, further truncation of this fragment (NsiI-HinfI) still results in binding of a protein by EMSA, but the complex is no longer hypoxia-inducible.
Genistein, a tyrosine kinase inhibitor, was recently shown to inhibit the hypoxic induction of VEGF mRNA (28) through its action on Src. A signal transduction cascade leading to the hypoxic induction of VEGF through Raf was demonstrated using the dominant inhibitory Raf-1 mutant Raf(301)(28, 29) . In other systems this cascade has been shown to proceed through mitogen-activated protein kinase and ultimately to modulate transcription of specific genes and phosphorylation of specific gene products(30, 31) . We have shown here that genistein interferes with the post-transcriptional induction of VEGF by hypoxia. 500 uM genistein had no effect on the hypoxic induction of 5` promoter reporter constructs but inhibited the preferential stabilization of VEGF 3`-UTR transcripts in vitro by hypoxic versus normoxic extracts and inhibited formation of a hypoxia-inducible protein-RNA complex by EMSA. One possibility is that the signal cascade through src/raf and possibly mitogen-activated protein kinase has as its terminal event a protein that binds to the VEGF mRNA 3`-UTR and mediates its hypoxic stabilization.
An understanding of the molecular basis of the regulation of VEGF by hypoxia forms the essential groundwork for the rational design of pharmacological agents to modulate VEGF expression and thereby augment or inhibit neovascularization. The in vitro degradation assays and EMSA described here should allow for the rapid and economic assessment of multiple agents that may affect VEGF mRNA stability.