From the Cardiology Division, Georgetown University
Medical Center, Washington, D. C. 20007 and the § Program
in Molecular Pharmacology and Therapeutics, Memorial Sloan Kettering
Cancer Center, New York, New York 10021
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ABSTRACT |
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Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is dramatically induced by hypoxia due in large part to an increase in the stability of its mRNA. Here we show that HuR binds with high affinity and specificity to the element that regulates VEGF mRNA stability by hypoxia. Inhibition of HuR expression abrogates the hypoxia-mediated increase in VEGF mRNA stability. Overexpression of HuR increases the stability of VEGF mRNA. However, this only occurs efficiently in hypoxic cells. We further show that the stabilization of VEGF mRNA can be recapitulated in vitro. Using an S-100 extract, we show that the addition of recombinant HuR stabilizes VEGF mRNA markedly. These data support the critical role of HuR in mediating the hypoxic stabilization of VEGF mRNA by hypoxia.
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INTRODUCTION |
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Vascular endothelial growth factor (VEGF)1 has been demonstrated both in vitro and in vivo to be a significant mediator of hypoxia-induced angiogenesis in such diverse disease processes as diabetic retinopathy (1), tumor angiogenesis (2), and coronary artery disease (3). Pharmacological manipulation of VEGF in these disorders either to augment or to inhibit neovascularization requires an understanding of the molecular mechanisms regulating VEGF induction by hypoxia.
We have demonstrated previously that the increase in VEGF protein and biological activity secreted by cells exposed to hypoxia is in large part the result of an increase in VEGF mRNA stability with the half-life of VEGF mRNA increasing 3-8-fold under hypoxic conditions (4-6). In vitro RNA degradation assays performed with VEGF mRNA and S-100 extracts from normoxic and hypoxic cells have permitted not only the identification of specific regions of VEGF mRNA which are responsible for the lability of VEGF mRNA under normoxic conditions but also regions that are critical for the stabilization of VEGF mRNA by hypoxia (5). In the case of the destabilizing elements in the VEGF 3'-untranslated region (UTR) the sequences correspond to canonical nonameric instability sequences (7-9) that have been shown to mediate the rapid turnover of many other cytokines and oncogenes.
With regard to the hypoxia-stabilizing element in the VEGF 3'-UTR we have described previously a novel hypoxia-inducible protein complex that binds to a adenylate-uridylate (AU)-rich element in this region (6). Affinity purification of the hypoxia-inducible protein complex using an RNA corresponding to this binding site in the VEGF mRNA 3'-UTR has allowed for the identification of three hypoxia-inducible proteins of approximately 34, 28, and 17 kDa.
The identity of these proteins has remained unknown. It is clear that at least one of the proteins in this complex binds specifically to an AU-rich element in the VEGF 3'-UTR. The cellular proteins that bind to such AU-rich elements have been the focus of much investigation (10-15). Recently, a 36-kDa RNA binding protein, HuR, which binds to AU-rich elements with high affinity and selectivity, has been identified and cloned (14, 16, 17). In view of its similarities in binding specificity and size to the 34-kDa component of the hypoxia-inducible protein complex, we sought to determine whether HuR might play a role in the hypoxic stabilization of VEGF mRNA.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Culture Conditions-- 293T cells were obtained from the ATCC. WT-8 (18) and 786-0 cells were obtained from Dr. William Kaelin (Dana Farber Cancer Institute, Boston, MA). All cell lines were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were cultured under either normoxic conditions (5% CO2, 21% O2, and balance N2) or hypoxic conditions (5% CO2, 1% O2, balance N2) in a Forma 3130 incubator (Forma Scientific, Marietta, OH).
Preparation of S-100 Extracts and in Vitro RNA Degradation Assays with HuR and HuR Antiserum-- The S-100 fraction of cytosolic proteins was prepared and in vitro RNA degradation assays performed as described previously (5).
Preparation of Labeled RNA Transcripts--
pSP65Hg (-globin)
was linearized with Sau3AI. This template was incubated with
Sp6 RNA polymerase and [32P]UTP yielding a transcript of
165 nucleotides. pSP65Hg was provided by Dr. Gary Brewer. The DNA
templates for the VEGF transcripts (fragment VRS and its subfragments
A-F) were synthesized by polymerase chain reaction using the following
oligonucleotides. For VEGF template VRS corresponding to VEGF 3'-UTR
nucleotides 1252-1878 the oligonucleotides were T71252
(CCTAATACGGACTCACTATAGGGAGAATTTCAACTATTTATGAGGA) and 1878a
(TTTGAGATCAGAATTCAATTCTTTAATACAAAATGCC). For subfragment A
corresponding to VEGF 3'-UTR nucleotides 1252-1470 the
oligonucleotides were T71252 and 1470a
(TTCAAAGGAATGTGTGGTGGGGAC). For subfragment B corresponding to VEGF
3'-UTR nucleotides 1472-1510 the oligonucleotides were
T71472
(CCTAATACGACTCACTATAGGGAGGTAAGGTTTCAATATACATTTACATAC) and
1510a (ACAAGTTGCCAAATATATATATAG). For subfragment C corresponding to VEGF 3'-UTR nucleotides 1508-1573 the oligonucleotides were T71508
(CCTAATTACGACTCAGTATAGGGAGTTTGGCAACTTGTGTTTGTATATAAA) and 1573a
(CAGAATCACATATATACATAAAC). For subfragment E corresponding to VEGF
3'-UTR nucleotides 1631-1678 the oligonucleotides were T71631 (TCCTAATACGACTCACTATAGGGAGAATTCTACATACTAAATCTCTCTCC)
and 1678a (CAAATATTAAAATTAAAAAAGGAGAGAGA). For subfragment
F corresponding to VEGF 3'-UTR nucleotides 1695-1878 the
oligonucleotides were T71695
(CCTAATACGACTCACTATAGGGAGGTGCTACTGTTTATCCGTAAT) and 1878a. All VEGF
templates were gel purified and RNA made using T7 RNA polymerase. All
transcripts were gel purified as described previously (17).
RNA Complex Assay--
Reaction mixtures (0.02 ml) contained 50 mM Tris (pH 7.0), 150 mM NaCl, 0.25 mg/ml tRNA,
0.025 mg/ml bovine serum albumin, 2.6 fmol of labeled RNA, and protein
as indicated. Mixtures were incubated at 37 °C for 10 min. After
incubation, 5 µl of a dye mixture (50% glycerol, 0.1% bromphenol
blue, 0.1% xylene cyanol) was added, and 5 µl of the mixture was
loaded immediately on a 0.8% agarose gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA). The gel was then
electrophoresed at 40 volts for 2.5 h. The gel was dried on DE81
paper (Whatman) with a backing of gel drying paper (Hudson City Paper,
West Caldwell, NJ) and exposed to XAR5 film (Eastman Kodak) for 6 h at 70 °C.
Nitrocellulose Filter Binding Assay-- Reaction mixtures (0.02 ml) contained 50 mM Tris (pH 7.0), 150 mM NaCl, 0.025 mg/ml bovine serum albumin, 0.25 mg/ml tRNA, 2.6 fmol of radiolabeled mRNA, and purified HuR as indicated. After 10 min of incubation at 37 °C, the mixtures were diluted 1:6 with buffer F (20 mM Tris (pH 7.0), 150 mM NaCl, 0.25 mg/ml tRNA) and filtered through nitrocellulose (BA85, Schleicher & Schuell). After washing the filter twice with buffer F, bound radioactivity was determined by Cerenkov counting.
RNase T1 Selection Assay--
Reaction mixtures (0.02 ml)
contained 50 mM Tris (pH 7.0), 150 mM NaCl,
0.25 mg/ml bovine serum albumin, 0.25 mg/ml tRNA, 80 fmol of
radiolabeled mRNA, and purified HuR as indicated. After 10 min of
incubation at 37 °C, RNase T1 was added, and the reaction continued
for a further 10 min. The mixtures were diluted 1:6 with buffer F and
filtered through nitrocellulose (BA85). After washing the
nitrocellulose twice with buffer F, bound HuR·RNA complex on
nitrocellulose filter was eluted with buffer F and extracted with
phenol-chloroform. The resultant RNA was mixed with formamide buffer,
denatured at 65 °C for 3 min, and analyzed by 12%
polyacrylamide/urea gel electrophoresis. The gel was fixed with 1:1:8
acetic acid:methanol:water, dried on DE81 paper with a backing of gel
drying paper, and exposed to XAR5 film at 70 °C overnight.
Establishment of Cell Lines Overexpressing Antisense HuR mRNA
or Sense HuR mRNA--
An 1.6-kilobase ApaI fragment of
HuR9 (17) (containing the entire coding sequence of HuR9) was cloned in
either the sense (H.26) or antisense (R.11) orientation in pZeoSV2()
(Invitrogen) and introduced into 293T cells by electroporation. Stable
transfectants were selected with Zeocin (Invitrogen). Zeocin clone mvr
29 was prepared by transfection of wild type 293T cells with the
pZeoSV2(
) vector backbone.
Affinity Purification of Anti-HuR Antibody--
An HuR affinity
column was prepared by conjugating GST-HuR to Affi-Gel 10 (5 mg of
HuR/ml of resin). A high titer anti-Hu serum (19) (obtained from Athena
Diagnostics) was applied to the column (10 ml of serum/ml of resin),
and the column was washed with buffer W (50 mM Tris-HCl (pH
7.5), 0.2 M NaCl, 0.01% Nonidet P-40). After extensive
washing the bound antibody was eluted with 0.1 M NaCl, 0.1 M sodium citrate (pH 2.5) and neutralized immediately with
Tris base. The eluate fractions were assayed by Western blot analysis
using recombinant HuR. Active fractions were pooled (typical concentration is 110 µg/ml IgG) and stored at 70 °C.
Western Blot of Total Cell Extract from 293T Clones with HuR Affinity-purified Antiserum-- For preparation of total cell extract from 293T clones mvr 29, H.26, and R.11 the cells were first washed twice with ice-cold phosphate-buffered saline and then lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 2 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin). Protein concentration of the extract was determined using the Bio-Rad protein assay. 10 µg of each extract was then boiled in SDS sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and then transferred to Immobilon polyvinylidene difluoride membrane (Millipore). Membrane was then blocked with 5% milk in TBS-Tween (150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 0.05% Tween 20). Human HuR affinity-purified antiserum (see above) was used at a concentration of 1.08 µg/ml followed by alkaline phosphatase-conjugated rabbit anti-human antibody (Promega).
Measurement of VEGF mRNA Half-life-- The half-life of VEGF mRNA was determined using actinomycin D as described previously (5). The quantity of VEGF mRNA was normalized to the amount of 18 S rRNA by calculating a VEGF:18 S ratio for each sample (5).
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RESULTS |
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HuR Specifically Binds to the Regulatory Element in the 3'-UTR of
VEGF mRNA--
Fig. 1 shows the
structure of VEGF mRNA and of the RNAs (VRS, A, B, C, E, and F)
used in these studies. VRS (the VEGF regulatory segment) contains the
signals necessary for the post-transcriptional regulation of VEGF
mRNA (5). Previous studies have shown that HuR binds specifically
to similar regulatory elements in other mRNAs (14, 17). Thus we
investigated whether HuR can bind to the regulatory elements of VEGF
mRNA. Purified recombinant GST-HuR fusion protein was incubated
with labeled transcripts and HuR·RNA complex formation assayed by gel
retardation analysis (17, 20). HuR bound avidly to the VRS, and complex
formation was easily detectable with 1 nM HuR (Fig.
2, lanes 5-8). No complex formation was detectable in the absence of HuR or with 200 nM GST. Complex formation is specific since no reaction was
observed with a control RNA (the 3'-end of the human -globin
mRNA) of similar composition which does not contain an AU-rich
regulatory element (Fig. 2, lane 2). The interaction between
HuR and the VRS was investigated further using a quantitative RNA
binding assay. We employed the same method as used originally for the R17 coat protein (21). A low concentration of labeled RNA was incubated
with increasing concentrations of HuR protein as indicated. The
reactions were filtered through nitrocellulose and the bound radioactivity determined. Fig.
3A shows that the formation of the VEGF mRNA·HuR complex is detectable at 0.1 nM,
has a midpoint at about 9 nM, and reaches a plateau above
50 nM with about 80% of the input RNA bound. Complex
formation with globin RNA was not detectable under these conditions
(Fig. 3A). A plot of the log of complex/free RNA
versus log of HuR concentration revealed a straight line
with an intersect on the x axis at 9 nM (Fig. 3B). Thus the binding of HuR to VEGF mRNA is a simple
molecular reaction with an apparent Kd of 9 nM.
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Antisense HuR mRNA Blocks the Hypoxic Stabilization of VEGF mRNA-- The in vitro binding data described above clearly demonstrate that HuR binds selectively to an AU-rich element in the regulatory element of VEGF mRNA. To determine if HuR plays an important role in the hypoxic regulation of VEGF mRNA stability in vivo, we next developed stable transformants of human 293T cells which expressed antisense HuR mRNA constitutively. 293T cells were chosen because the hypoxic induction of VEGF mRNA (2.4 ± 0.3-fold) in 293T cells is due solely to post-transcriptional mechanisms with no hypoxic induction of reporter elements that have been shown to mediate the hypoxic induction of genes such as erythropoietin and VEGF (23-25).
Antisense HuR under the control of the cytomegalovirus promoter was cloned into the pZeoSV2(
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Overexpression of HuR mRNA Increases the Stability of VEGF mRNA under Hypoxic Conditions-- Although the antisense studies described above demonstrate that HuR is critical for the hypoxic stabilization of VEGF mRNA they do not address whether HuR is sufficient to increase VEGF mRNA stability. We therefore sought to determine if overexpression of HuR could increase VEGF mRNA in vivo by developing stable transformants of human 293T cells which constitutively overexpressed HuR.
HuR under the control of the cytomegalovirus promoter was cloned into the pZeoSV2(
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Purified HuR Stabilizes VEGF mRNA in an in Vitro RNA Degradation Assay-- We have described previously a specific RNA in vitro degradation system using capped, polyadenylated VEGF 3'-UTR transcripts and S-100 cytoplasmic extracts (5). We believe that this in vitro system recapitulates regulated expression faithfully since RNA degradation is dependent on an AU-rich element and reflects the oxygen environment of the cells before the preparation of the extract. Thus we investigated whether the in vitro system would respond to exogenously added HuR. Extracts were prepared from normoxic WT-8 cells (18). Fig. 7 shows that VEGF mRNA decays quickly with a half-life of 8.5 min. The addition of HuR, however, stabilized VEGF mRNA markedly with an increase in the half-life to 30 min.
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DISCUSSION |
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The increase in the stability of VEGF mRNA in response to a reduction of the oxygen tension of the cellular milieu has been demonstrated to be mediated by specific sequences in its 3'-UTR. A clearer understanding of the mechanism of this regulation requires the identification and characterization of the transacting factors that interact with these elements. This work identifies HuR as one of the important transacting factors that bind selectively to these sequences and thereby regulate VEGF expression. HuR is a member of the Elav-like protein family. Elav, the founder member of this family, is a Drosophila RNA-binding protein required for neuronal differentiation (27). Interest in this family of proteins was originally fueled by the observation that their human homologs are tumor antigens (19). There are four members of the human Elav-like family, namely, HuD, HuC, Hel-N1, and HuR (17, 19, 28, 29). HuD, HuC, and Hel-N1 are expressed exclusively in postmitotic neurons and in specific neuroendocrine tumors (29-32). HuR, the fourth and newest member, is transcribed in all cells (17). The unique structural feature of the Elav-like proteins is the organization of their three ribonucleoprotein-2/ribonucleoprotein-1 type RNA recognition motifs (33). The first and second of these RNA recognition motifs are in tandem and are separated from the third by a segment rich in basic amino acids. The observation that these motifs bind selectively to the AU-rich elements that regulate mRNA turnover suggested that the Elav-like proteins are involved in the post-transcriptional control of gene expression (17, 20, 29, 34, 35). The results presented here on HuR and those recently obtained with HuD2 and Hel-N1 (36) confirm and extend this hypothesis.
Our data indicate that HuR is necessary for the post-transcriptional induction of VEGF expression. Inhibition of HuR expression by antisense constructs inhibits stabilization of VEGF mRNA by hypoxia. On the other hand our data do not permit us to conclude whether or not overexpression of HuR is sufficient for the stabilization of VEGF mRNA. Our data demonstrate that HuR protein was increased to a greater extent under hypoxic conditions than under normoxic conditions. We cannot rule out the possibility that the failure to stabilize VEGF mRNA in the overexpressing clones under normoxic conditions is due to an inadequate amount of HuR protein to mediate this effect. Thus we cannot conclude from these studies whether or not the stabilization of VEGF mRNA by hypoxia requires factors in addition to HuR which are also induced by hypoxia.
Why is HuR critical for the hypoxic stabilization of VEGF mRNA? Our
data indicate that total cellular steady-state HuR protein is unchanged
by hypoxia. This may suggest that HuR is a critical component of a
hypoxia-inducible complex (6) whose other components are regulated by
hypoxia. Such a precedent has been set recently for the
hypoxia-inducible transcription factor HIF-1 (37-39). HIF-1 is a dimer
of HIF-1, which is regulated by hypoxia (39), and HIF-1
, which is
not regulated by hypoxia. Nonetheless, elimination of cellular HIF-1
activity blocks the ability to transactivate hypoxia-inducible genes
that are dependent on HIF-1 for this induction (38).
On the other hand, we have not measured directly the amount of active HuR in hypoxic and normoxic cells. Conceivably, HuR protein could be localized differentially in the cell under hypoxic or normoxic conditions, an intriguing possibility given the known ability of many such RNA-binding proteins to shuttle between the nucleus and the cytoplasm (40). Alternatively, HuR may be regulated by phosphorylation.
The observation that the Elav-like proteins stabilize mRNA that contains AU-rich elements has important implications for the understanding of the mechanism of mRNA turnover. The accepted model is that the AU-rich elements are recognized by specific factors. In some models the AU-rich element is recognized by a specific endonuclease. Cleavage of the transcript then occurs followed by rapid degradation catalyzed by a 3'-5' exonuclease (9, 41). In other models it is proposed that the AU-rich element promotes the deadenylation of the mRNA. Deadenylation of the transcript is again followed by rapid 3'-5' exonuclease degradation. In either case, it has been proposed that stability factors bind to the AU-rich element and inhibit either the endonuclease activity (42) or the deadenylase activity. In this paper we have mapped the binding site of HuR to be identical to the hypoxia-inducible complex RNA binding site. HuR binds within four nucleotides of a canonical nonameric instability element (7, 8) in the VEGF AU-rich element. Thus it is likely that HuR binding alters the structure of the AU-rich element and renders the site either inaccessible to the endonuclease or unable to promote deadenylation. Our ability to derive an in vitro system that responds to the addition of HuR will facilitate the purification of the putative factors and will permit a detailed examination of the mechanism of this reaction.
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FOOTNOTES |
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* This work was supported by Grants NS29682 (to H. F.) and 1K08HL03405-02 (to A. P. L.) from the National Institutes of Health, a grant from the Byrne fund (to H. F.), Core Grant P30-CA08748 from the NCI, National Institutes of Health, and a grant-in-aid from the American Heart Association (to A. P. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Haifa, Israel 31096. To whom correspondence should be sent. Tel.: 972-4-829-5202; Fax: 972-4-852-0089; E-mail: alevy{at}tx.technion.ac.il.
1 The abbreviations used are: VEGF, vascular endothelial growth factor; UTR, untranslated region; AU, adenylate-uridylate; VRS, VEGF regulatory segment; GST, glutathione S-transferase; HIF, hypoxia-inducible factor.
2 G. B. Aranda-Abreu, S. Chung, H. Furneaux, and I. Ginzburg, submitted for publication.
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REFERENCES |
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