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J. Biol. Chem., Vol. 277, Issue 16, 13635-13640, April 19, 2002

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A 40-bp RNA Element That Mediates Stabilization of Vascular Endothelial Growth Factor mRNA by HuR^*

Ilana Goldberg-Cohen, Henry Furneauxb^§, and Andrew P. Levy^¶

From the  Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Post Office Box 9649, Haifa 31096, Israel and the ^§ Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06032

Received for publication, September 10, 2001, and in revised form, January 17, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

VEGF is a critical mediator of hypoxia-induced angiogenesis in numerous physiological and pathophysiological conditions. The hypoxic induction of VEGF is due in large part to an increase in the stability of its mRNA. We recently demonstrated that the stabilization of VEGF mRNA by hypoxia is dependent upon the RNA-binding protein HuR. This report describes the identification of a 40-bp functional HuR binding site in the VEGF mRNA 3'-untranslated region. This element can confer HuR-mediated stabilization of a heterologous gene in vitro and in vivo. Furthermore, the element is sufficient to confer an increase in the hypoxic induction of a heterologous gene. Deletion of the HuR binding site within this 40-bp element as mapped by RNase T1 and lead footprinting uncouples a stabilizing sequence from a destabilizing sequence, thus providing a novel RNA-protein regulatory model that might be exploited to manipulate VEGF expression and hypoxia-induced angiogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vascular endothelial growth factor (VEGF)¹ has been demonstrated both in animal models and in man 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). A rational pharmacological approach to manipulate VEGF in these disorders either to augment or inhibit neovascularization requires an understanding of the molecular mechanisms regulating the hypoxic induction of VEGF.

We and others (4-10) have previously demonstrated 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 severalfold under hypoxic conditions. In vitro RNA degradation assays performed with VEGF mRNA and S-100 extracts from normoxic and hypoxic cells have permitted the identification of sequences that are critical for the hypoxic stabilization of VEGF mRNA (5). Affinity purification of a hypoxia-inducible RNA binding complex using these sequences has revealed multiple proteins that can bind to this region (6, 10, 11). We subsequently demonstrated that one of these proteins, HuR (12), is critical for the hypoxic stabilization of VEGF mRNA by inhibiting the hypoxic stabilization of VEGF mRNA via overexpression of antisense HuR (13).

The minimal sequences in VEGF mRNA necessary for HuR to bind and stabilize the RNA are not known. We sought to identify a functional HuR binding site in the VEGF mRNA and to determine whether this minimal element could confer HuR-mediated stabilization and hypoxia-inducible stabilization in a heterologous context.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines and Culture Conditions-- 293 cells were obtained from the ATCC. 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, balance N₂) or hypoxic conditions (5% CO₂, 1% O₂, balance N₂) in a Jouan 750 triple gas incubator.

Preparation of S-100 Extracts and in Vitro RNA Degradation Assays with HuR-- The S-100 fraction of cytosolic proteins was prepared as described previously (5). DNA templates containing the 40-bp element of the VEGF mRNA 3'-untranslated region (nucleotides 1285-1325 of the VEGF 3'-UTR, GenBank^TM accession number U22372) inserted into the 3'-untranslated region of the luciferase gene were prepared as follows. For preparation of the wild type 40-bp element, overlapping oligonucleotides designed to be flush at the 5' end and possessing a SacI overhang at the 3' end were 1285s, 5'-CTTTCTTATTTGTACTGTTTTTTTTTTTTGTTTTGTTTTTACTAGTCGAGCT-3', and 1285as, 5'-CGACTAGTAAAAACAAAACAAAAAAAAAAAACAGTACAAATAAGAAAG-3'. For preparation of the deletion mutant of the 40-bp element, overlapping oligonucleotides designed to be flush at the 5' end and possessing a SacI overhang at the 3' end were 1285ms, 5'-CTTTCTTATTTGTACTGTTTTTTTACTAGTCGAGCT-3', and 1285mas, 5'-CGACTAGTAAAAACAGTACAAATAAGAAAG-3'. The oligonucleotides were phosphorylated, annealed, and then ligated directly into pGEM-luc (Promega) at the StuI-SacI sites. Capped ³²P-labeled transcripts from the linearized pGEM-luc template with or without the 40-bp element (pGEM-luc40 or pGEM-luc) were made using SP6-Megascript (Ambion) reagents and were used in the in vitro RNA degradation assays as described previously (5). For all degradation assays, a direct comparison of the stability of the luc or luc40 RNA with an equivalent amount of GST-HuR or GST protein (final concentration of 5-500 nM) was performed. GST-HuR or GST was added to the extracts prior to their incubation with the labeled RNA.

Transient Transfection Assay with HuR and Luc Reporter Containing VEGF Sequences in the 3'-UTR-- 293 cells were cotransfected with a plasmid overexpressing HuR (13) and a luciferase reporter plasmid containing VEGF mRNA sequences in the 3'-UTR of luciferase. Transfection was performed by electroporation using a BTX ECM 600 electroporator with the following settings (800 microfarads of resistance setting 4; 270 V). Electroporation was performed with 4-mm cuvettes with 1 µg of salmon sperm DNA, plasmid DNA (total of 5 µg), and 4 × 10⁶ cells in 1000 µl of Dulbecco's modified Eagle's medium with 10% fetal bovine serum.

The HuR plasmid was described previously (13) and consists of a 1.6-kb Apa fragment of the HuR cDNA cloned into pZeoSV2(-) (Invitrogen). The backbone luciferase vector was pxp2 (14) (ATCC). For all studies, the luciferase vector also contained 1.7 kb of VEGF promoter sequence (vector 1.7pxp as described previously (4)) cloned 5' to the luciferase gene. Fragments of the VEGF mRNA were generated by restriction endonuclease digestion or by PCR and cloned into 1.7pxp. Vector pxp53 contains the entire VEGF 5'-UTR cloned 5' to the luciferase gene and nucleotides 1-1751 of the VEGF 3'-UTR cloned 3' to the luciferase gene. Vector pxp8a contains nucleotides 1-1751 of the VEGF 3'-UTR inserted in the 3'-UTR of luciferase in 1.7pxp. Vectors pxpNsi, pxp1695, pxp1580, and pxp1400 represent fragments of the VEGF 3'-UTR corresponding to sequences 1255-1751, 1695-1751, 1255-1580, and 1255-1400 (GenBank^TM accession number U22372) cloned 3' to the luciferase gene in 1.7pxp. Vector 1.7pxp40M, containing a 16-bp deletion of 1.7pxp40, was generated using oligomers 1285ms and 1285mas as described above. For all vectors, the polyadenylation signal sequence for the heterologous luciferase transcript was supplied by SV40 sequences located 3' to the VEGF sequences. In addition, all vectors contained intronic sequences supplied by SV40 sequences located 5' to the polyadenylation signal sequence. Both the intronic sequences and polyadenylation signal sequence were those present in the pxp2 backbone as described originally (14). For each cotransfection experiment, 1 µg of luciferase reporter plasmid was cotransfected with a 0.05-5 ng of HuR plasmid. Luciferase activity was determined in cells cultured under normoxic conditions 24 h after transfection using a TD 20/20 luminometer and Luc pack reagents (both from Promega).

Transient Transfection of VEGF Reporter Plasmids under Normoxic and Hypoxic Conditions-- VEGF reporter plasmids were prepared, and electroporation was performed in 293 cells as described above using 1 µg of reporter plasmid. After electroporation, half of the cells were placed under hypoxic conditions, and half were placed under normoxic conditions. Luciferase activity was measured after 24 h of hypoxia. Luciferase activity was normalized to total protein concentration in the cell lysate from transfected cells. All transfections were done in triplicate or quadruplicate. Each transfection was repeated at least five times.

Footprint Analysis of HuR Binding to the 40-bp Element-- A 47-base RNA oligonucleotide of sequence 5'-CUUUCUUAUUUGUACUGUUUUUUUUUUUUGUUUUGUUUUUCUGUGUG-3' was synthesized and used for HuR binding studies. RNase T1 and lead protection mapping were performed as described previously (15).

Statistical Analysis-- All values are reported as the mean ± S.E. with the number of replicates given for each experiment. Comparisons between groups were performed using Student's t test. This test was two-tailed with a significance level of p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HuR Regulation of Reporter Gene Activity-- We have previously demonstrated that recombinant HuR protein stabilizes VEGF mRNA in an in vitro RNA degradation assay (13). We sought a more rapid and simpler method to map regions of the VEGF mRNA that are important for HuR-mediated changes in stability. Accordingly, we developed a transient cotransfection assay with one plasmid overexpressing HuR protein and the second plasmid consisting of a luciferase gene in which wild type luciferase mRNA 3'-UTR sequences were replaced by those from VEGF mRNA. We generated a variety of luciferase constructs containing various portions of the VEGF 3'-UTR as outlined under "Experimental Procedures" and Fig. 1. No increase in reporter activity was seen using luciferase constructs lacking VEGF mRNA 3'-UTR sequences (vector 1.7pxp) in these cotransfection experiments over a wide range of HuR plasmid concentrations. However, we found a statistically significant 2-fold HuR-inducible increase in luciferase activity in contransfection studies using reporter constructs containing the entire VEGF 3'-UTR as compared with constructs lacking VEGF sequences. Progressive deletion analysis of the VEGF 3'-UTR localized a sequence between nucleotides 1250 and 1400 of the VEGF 3'-UTR, which could confer a statistically significant HuR-mediated increase in reporter activity (Fig. 1). Within this region, we identified an adenylate-uridine-rich sequence, which we predicted would bind to HuR based on the reported high affinity binding of HuR to such sequences (12, 13, 15). A 40-bp fragment containing this sequence was found to confer a statistically significant HuR-inducible increase in reporter activity in the cotransfection assay. Further characterization of the HuR dose dependence of this enhancement of reporter gene activity by this 40-bp element revealed a relatively narrow window of cotransfected HuR in which luciferase reporter activity was increased (Fig. 2a). Deletion of the HuR binding site within this 40-bp element in the reporter construct 1.7pxp40M eliminated the ability of cotransfected HuR to increase reporter activity (Fig. 2b).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Mapping of a HuR responsive element. pxp2 luciferase VEGF mRNA heterologous constructs were prepared as described under "Experimental Procedures" and shown schematically here. For each luciferase construct, cotransfection with pSV2Zeo(-) overexpressing HuR was performed using a range of HuR plasmid concentrations (0.05-5 ng) and a fixed amount of luciferase reporter plasmid (1 µg) in 293 cells. The mean-maximal -fold induction of luciferase activity for a given construct with cotransfection of the HuR plasmid compared with the absence of any transfected HuR plasmid is shown. For all constructs, transfection was repeated at least five times. For all constructs, a statistically significant increase in HuR-inducible luciferase activity is noted (asterisk) if p < 0.05, comparing the values obtained with and without HuR cotransfection. HuR cotransfection did not increase reporter gene activity for 1.7pxp, pxp1.740M, or for 1.7pxp-containing VEGF mRNA 3'-UTR sequences 1695-1751 placed in the 3'-UTR of luciferase at any concentration of the HuR expression plasmid that was used in the cotransfection studies.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Dose-dependent increase in luciferase reporter activity. Data shown is for luciferase reporter 1.7pxp40 (a) or 1.7pxp40M (b) containing either the wild type and mutant 40-bp element in the 3'-UTR of luciferase. Total amount of cotransfected HuR expression plasmid is shown. Luciferase activity is expressed as the ratio of that obtained without transfection of the HuR plasmid. There was a significant HuR-inducible increase in reporter activity using 1.7pxp40 (maximal induction, 1.7 ± 0.3, p < 0.05, n = 6) but not with 1.7pxp40M. This increase was seen over a narrow window of the cotransfected HuR plasmid.

HuR Protein Binds to the 40-bp Minimal Element in the VEGF 3'-UTR-- A 47-mer RNA oligonucleotide containing the 40-bp sequence necessary to obtain a HuR-mediated enhancement in reporter activity is shown in Fig. 3a. The binding site for HuR within this sequence was mapped by lead protection and RNase T1 shown schematically in Fig. 3a. RNase T1 mapping indicated that the HuR binding site was between G17 and G43 (Fig. 3b). Lead normally cleaves RNA at all residues, and thus lead protection is a more definitive determination of the site of binding of a RNA-binding protein. The extent of protection by HuR against hydrolysis with lead permitted the identification of a putative HuR binding site between residues U23 and U39 (Fig. 3c).

View larger version (65K): [in this window] [in a new window]   Fig. 3.   Mapping of the HuR binding site by lead and RNase T1 protection. a, the sequence of the 47-base RNA oligonucleotide used for these studies and the putative HuR binding site is shown. The arrows indicate residues that are hyperhydrolyzed by lead indicating the extent of HuR protection. b, RNase T1 footprint analysis of the HuR binding site. Reaction mixtures (10 µl) contained 50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 50 µg/ml tRNA, 3 nM labeled RNA, and purified GST or GST-HuR as indicated (concentrations are nM). Mixtures were incubated at 37 °C for 10 min, RNase T1 was then added to a final concentration of 0.05 units/ml, and the mixture further incubated for 10 min at 4 °C. Reactions were then terminated, and the reaction was analyzed by 10% polyacrylamide/urea gel electrophoresis. RNase T1 mapping indicated that the binding site for HuR was between G17 and G43, because G30 and G35 were protected from RNase attack. c, lead footprint analysis of the HuR binding site. Reactions were conducted as described above with the exception that lead acetate (10 mM) was substituted for RNase T1 after the preincubation. A RNase T1 digest of the element was run in a parallel lane to provide size markers as denoted on the left-hand side of the figure. Hyperhydrolyzed residues, indicated by arrow in a and seen in the figure as enhanced degradation in the presence of HuR, indicate the boundary of the extent of HuR binding and protection (i.e. U40, C41, and U42). Hypohydrolyzed residues, seen in the figure as decreased degradation in the presence of HuR, are residues U23-U39.

The 40-bp Element Confers Increased Stability to a Heterologous Transcript in Vitro-- Despite the previously reported role of HuR in mediating stabilization of a variety of mRNA (16-22), the increase in luciferase activity in the presence of the 40-bp element in the cotransfection studies could be explained by changes in the transcription of the luciferase gene or translation of the luciferase mRNA. Therefore, we tested the stability of luciferase RNA with or without this 40-bp element in the in vitro RNA degradation assay. Capped ³²P-labeled luciferase RNA was made by SP6 polymerase in vitro from DNA templates with or without the 40-bp element located in the luciferase 3'-UTR. GST-HuR or GST was added to the extracts prior to commencing the RNA degradation assays. At a final concentration of 50 nM HuR-GST or 50 nM GST, the luc transcript lacking the 40-bp element, luc RNA, was not preferentially stabilized by HuR as compared with GST (Fig. 4, a and c). However, we observed that the luciferase RNA containing the 40-bp element, luc40 RNA, was approximately twice as stable with 50 nM HuR-GST as compared with 50 nM GST (Fig. 4, b and c). Therefore, the stability conferred by HuR in vitro to luciferase RNA was dependent on this 40-bp site being present in the RNA. The concentration of HuR used in these studies is physiologically consistent with the known K_d of HuR for VEGF mRNA (13) and the estimated concentration of HuR in the cell. This preferential stabilization of luc40 RNA by HuR as compared with GST-HuR was not seen at a concentration of 5 nM HuR and GST (Fig. 4c).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Relative stability of luciferase RNA in an in vitro degradation assay in the presence of HuR-GST or GST alone. RNA degradation assays were performed as described under "Experimental Procedures." a, representative degradation assay using capped pGEM-luciferase RNA without the 40-bp RNA element, pGEM-luc, using 50 nM GST or 50 nM HuR-GST. The primary undegraded transcript that was used for quantitation of the stability of each transcript in the presence of HuR or GST is shown by an arrow. b, representative degradation assay using capped pGEM-luciferase RNA with the 40-bp RNA element, pGEM-luc40, using 50 nM GST (lower panel) or 50 nM HuR-GST (upper panel). The primary undegraded transcript that was used for quantitation of the stability of each transcript in presence of HuR or GST is shown by an arrow. c, histogram showing relative stability of luc or luc40 RNA in the presence of HuR-GST as compared with GST. Statistically significant enhancement of the stability of luc40 RNA by HuR as compared with GST was found at a concentration of 50 nM in this assay (p < 0.05 for comparing the ratio of the stability of luc40 RNA in the presence of HuR or GST to the ratio of the stability of luc RNA in the presence of HuR or GST at a concentration of GST or HuR of 50 nM). Data are the mean ± S.E. of five independent degradation assays each of which was performed in triplicate.

The 40-bp Element Acts in a Heterologous Context to Promote an Increase in Reporter Gene Activity under Both Normoxic and Hypoxic Conditions-- Because HuR is critical for the stabilization of VEGF mRNA and binds to this 40-bp element, we sought to determine whether the element could mediate an increase in gene expression in a heterologous context under both normoxic and hypoxic conditions. Accordingly, we transfected 293 cells with 1.7pxp and 1.7pxp40 and found that the level of reporter gene activity was significantly higher both under normoxic and hypoxic conditions by virtue of having this element (Fig. 5, a and b). Moreover, the hypoxic -fold induction of luciferase activity (ratio of luciferase obtained under hypoxia to normoxia) was significantly greater for reporter constructs containing the 40-bp element as opposed to constructs without the element (2.0 ± 0.1 versus 1.5 ± 0.1, p < 0.01).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Regulation of reporter gene expression under normoxic and hypoxic conditions with the wild type and mutant 40-bp element. Transfection of 293 cells by 1.7pxp, 1.7pxp40, and 1.7pxp40M was performed as described under "Experimental Procedures," and cells were cultured under either normoxic (a) or hypoxic (b) conditions. Transfections were performed in quadruplicate, and results are reported as the mean ± S.E. of transfections performed on five separate occasions. Data are reported as the mean ± S.E. There was a statistically significant increase in reporter activity for 1.7pxp40 compared with 1.7pxp under both normoxic and hypoxic conditions (p < 0.05 and p < 0.02, respectively) as well as a statistically significant increase in the hypoxic -fold induction for 1.7pxp40 compared with 1.7pxp as noted in the text. There was a statistically significant decrease in reporter activity for 1.7pxp40M compared with 1.7pxp under both normoxic and hypoxic conditions (p < 0.05 and p < 0.01).

Reporter construct 1.7pxp40M in which the HuR binding site within the 40-bp element was deleted demonstrated a marked significant decrease in reporter gene activity (both compared with 1.7pxp40 and 1.7pxp) under both normoxic and hypoxic conditions, suggesting that the residual sequence (i.e. the 40-bp element without the 16-bp HuR site) was destabilizing the reporter gene (Fig. 5, a and b). The hypoxic -fold induction (ratio of luciferase activity under hypoxic conditions to normoxic conditions) of 1.7pxp40M was significantly diminished as compared with 1.7pxp40 (1.5 ± 0.1 versus 2.0 ± 0.1, p < 0.01). Therefore, the deletion of the HuR binding site appears to have uncoupled a HuR and hypoxia-inducible RNA element from a destabilizing element.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have identified a 40-bp RNA element from the VEGF 3'-UTR mRNA that can confer a HuR-mediated increase in RNA stability and an increase in expression of a heterologous gene under both normoxic and hypoxic conditions. Moreover, we have uncoupled stabilizing and destabilizing sequences within this 40-bp element. Current paradigms have conceptualized that RNA proteins, which function to stabilize RNA, do so by binding to the identical sequences, which otherwise confer instability (15). Our studies suggest an alternative model in which RNA-stabilizing factors may bind to a distinct site and thereby change the secondary or tertiary structure of the RNA, rendering it inaccessible to instability factors and endonucleolytic attack by ribonucleases. The reporter constructs and cotransfection system we have described here are well suited for studying potential RNA-protein interactions and the stability and/or instability of mRNA in intact cells. This system could be used to design specific agents (i.e. decoy oligonucleotides) (11, 23) that may enhance or inhibit the interaction of HuR with its binding site and thereby increase or decrease VEGF mRNA expression. Furthermore, the identification of a RNA sequence, which can confer increased gene expression on a heterologous gene, is of tremendous importance in present technologies designed to deliver trans-genes (24-27). For example, this element could be incorporated into vectors designed to express angiogenic growth factors specifically in hypoxic regions of the myocardium (28).

We and others (13, 15, 29-31) have previously reported that HuR stabilizes RNA by binding to the body of the RNA and preventing endonucleolytic attack of the RNA. However, it is unclear why HuR binding might be important in the hypoxic induction of VEGF (i.e. more stability under hypoxic conditions as compared with normoxic conditions) (13). There is no apparent change in total HuR protein with hypoxia. However, HuR can shuttle back and forth from the nucleus to the cytoplasm (32), and hypoxia appears to promote an increase in the export of HuR from the nucleus to the cytoplasm.² HuR might then bind to VEGF mRNA in the nucleus and promote an increase in VEGF mRNA export under hypoxic conditions.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant RO1HL58510 (to H. F., and 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.

^¶ To whom correspondence should be addressed. Tel.: 972-4-8295202; Fax: 972-4-8514103; E-mail: alevy@tx.technion.ac.il.

Published, JBC Papers in Press, February 7, 2002, DOI 10.1074/jbc.M108703200

² A. P. Levy, unpublished data.

	ABBREVIATIONS

The abbreviations used are: VEGF, vascular endothelial growth factor; UTR, untranslated region; luc, luciferase; GST, glutathione S-transferase; luc40 RNA, luciferase RNA containing the 40-bp element; pGEM-luc40, pGEM-luciferase RNA with the 40-bp RNA element.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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