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Originally published In Press as doi:10.1074/jbc.M411275200 on March 23, 2005

J. Biol. Chem., Vol. 280, Issue 21, 20331-20339, May 27, 2005
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The Oxidative Stressor Arsenite Activates Vascular Endothelial Growth Factor mRNA Transcription by an ATF4-dependent Mechanism*

C. Nathaniel Roybal, Lucy A. Hunsaker, Olena Barbash, David L. Vander Jagt, and Steve F. Abcouwer{ddagger}

From the Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131-0001

Received for publication, October 4, 2005 , and in revised form, March 23, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aberrant retinal expression of vascular endothelial growth factor (VEGF) leading to neovascularization is a central feature of age-related macular degeneration and diabetic retinopathy, two leading causes of vision loss. Oxidative stress is suggested to occur in retinal tissue during age-related macular degeneration and diabetic retinopathy and is suspected in the mechanism of VEGF expression in these diseases. Arsenite, a thiol-reactive oxidative stressor, induces VEGF expression by a HIF-1{alpha}-independent mechanism. Previously, we demonstrated that homocysteine, an endoplasmic reticulum stressor, increases VEGF transcription by a mechanism dependent upon activating transcription factor ATF4. Because ATF4 is expressed in response to oxidative stress, we hypothesized that ATF4 was also responsible for increased VEGF transcription in response to arsenite. We now show that arsenite increased steady state levels of VEGF mRNA and activated transcription from a VEGF promoter construct. Arsenite induced eIF2{alpha} phosphorylation, resulting in increased ATF4 protein levels. Inactivation or loss of ATF4 greatly diminished the VEGF response to arsenite treatment. Overexpression of ATF4 was sufficient to activate the VEGF promoter, and arsenite cooperated with exogenous ATF4 to further activate the promoter. A complex containing ATF4 binds a DNA element at +1767 bp relative to the VEGF transcription start site, and DNA binding activity is increased by arsenite treatment. In addition, the ability of a thiol antioxidant, N-acetylcysteine, to inhibit the effect of arsenite on VEGF expression coincided with its ability to inhibit phosphorylation of eIF2{alpha} and ATF4 protein expression. Thus, arsenite-induced up-regulation of VEGF gene transcription occurs by an ATF4-dependent mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress is involved in the pathogenesis of two leading causes of vision loss, age-related macular degeneration (AMD)1 and diabetic retinopathy (DR). This association is indicated by the beneficial effects of antioxidant supplementation in AMD patients (1) and in streptozotocin diabetic rats (2). Furthermore, the breakdown of retinal antioxidant defenses has been demonstrated in aged and diabetic retinas (3, 4). However, the mechanism by which oxidative stress may contribute to the development of AMD and DR is still unknown.

Intraocular neovascularization is associated with both AMD and DR and is often a causative factor in the loss of vision during these pathologies. The neovascularization of the retina is frequently associated with increased expression of vascular endothelial growth factor (VEGF), a positive regulator of angiogenesis (for review, see Ref. 5). Retinal pigment epithelial cells are a major source of VEGF in diseased retinas (6, 7). However, the signaling mechanisms resulting in aberrant VEGF expression in aged and diabetic retinas are yet to be determined.

Oxidative stressors, hydrogen peroxide and superoxide, are positive regulators of VEGF in the retinal pigment epithelial cells (8). Antioxidant supplementation lowered VEGF protein levels in the retinas of diabetic rats (2). Arsenite, an oxidative stressor, was shown previously to up-regulate VEGF mRNA and hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) protein (9). This led to the suggestion that HIF-1 was responsible for VEGF induction by arsenite. However, further investigation demonstrated that arsenite failed to induce a functional HIF-1 complex and that the VEGF induction was HIF-1-independent (10). Therefore, an alternative pathway must regulate the induction of VEGF by arsenite.

Previously, we reported that homocysteine, a known ER stressor (11), which is elevated in DR (12), up-regulated VEGF expression through an endoplasmic reticulum stress response pathway that was dependent on activating transcription factor 4 (ATF4) (13). ATF4 protein levels increase in response to cellular stresses that cause global protein synthesis to be diminished. The ATF4 mRNA contains small upstream open reading frames that allow the ORF encoding ATF4 to be selectively translated when the translation initiation factor eIF2{alpha} is phosphorylated and levels of ternary initiation complex are low (14, 15). Activation of kinases that phosphorylate eIF2{alpha} leads to inhibition of global translation under conditions that perturb nascent protein processing, such as ER stress, nutrient deprivation, or exposure to oxidants and reactive metals (14, 16). ATF4 is a key mediator in the adaptive response of cells to oxidants. Import of the amino acids glycine and cysteine, precursors for glutathione biosynthesis, is impaired in ATF4-null cells (17). Other ATF4-inducible genes are mediators of the cytoprotective program activated by oxidative stressors, an example being heme oxygenase-1 (HO-1) (18).

Our previous findings implicating ATF4 in VEGF expression along with the current understanding of ATF4 as a regulator of redox homeostasis led us to question the role of ATF4 in the arsenite-mediated up-regulation of VEGF. We now demonstrate that arsenite induces eIF2{alpha} phosphorylation and that ATF4 protein up-regulation precedes VEGF mRNA up-regulation. ATF4 is necessary for VEGF mRNA up-regulation and for transcriptional activation of the VEGF promoter in response to arsenite. N-Acetylcysteine (NAC), which blocks the effect of arsenite on VEGF expression, also inhibited eIF2{alpha} phosphorylation and ATF4 protein expression. These results suggest a model wherein oxidative stress induces VEGF in an ATF4-dependent mechanism thereby contributing to the neovascularization seen in retinal pathologies such as AMD and DR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—ARPE-19 cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium (low glucose formulation) supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Immortalized mouse embryo fibroblasts (MEFs) were obtained from homozygous and heterozygous ATF4 knock-out MEF cultures (19) and were maintained in Dulbecco's modified Eagle's medium (high glucose formulation) supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 4 mM glutamine, and 50 µM thioglycerol. For Northern blotting experiments, cells were plated in 60-cm2 tissue culture dishes and grown to confluence. One day prior to being treated, ARPE-19 cells were fed with fresh media. MEF cultures were fed with fresh media containing 5 µM thioglycerol 1 day prior to treatment. Sodium arsenite (NaAsO2), N-acetylcysteine (NAC), ascorbic acid, trolox, and catalase (Sigma) were prepared fresh in Dulbecco's modified Eagle's medium and sterilized by filtration before being added to the cell cultures. For experiments utilizing adenoviral vectors, subconfluent cultures of ARPE-19 cells were treated with either ATF4 wild type (ATF4 Wt), ATF4 dominant negative mutant (ATF4 DN), or the empty AdEasy vector (Empty) (13). The ATF4 DN mutant protein contains a 6-amino acid substitution in the DNA-binding domain (292RYRQKKR298 to 292GYLEAAA298) (18). Twenty h post-infection, the percentage of cells infected was determined by examining the expression of enhanced green fluorescent protein by fluorescent microscopy.

Northern Blot Analysis—RNA isolation and Northern blotting was performed as described previously (20) using cDNAs corresponding to human VEGF (GenBankTM accession number dbEST189750), GRP78 (HAEAC89, ATCC), and heme oxygenase-1 (21) and normalized to 18 S rRNA. Rat heme oxygenase-A (GenBankTM accession number AA874884 [GenBank] ) was used for Northern analysis of MEF RNA. 18 S rRNA template was generated using mouse RNA as described previously (13). Membranes were hybridized with radiolabeled DNA probe for 6–8 h at 60 °C in a solution containing 7% (v/v) SDS, 0.25 M Na2HPO4, pH 7.2, as described (22). Blotting results were quantified by overnight exposure to a phosphor screen followed by analysis using a STORMTM PhosphorImager and ImageQuantTM software (Amersham Biosciences). For each sample, hybridization to 18 S rRNA was used to normalize results for mRNAs. Fold inductions were determined by dividing normalized mRNA band intensity volumes for experimental samples to that of control (untreated or time 0) samples.

VEGF Promoter Cloning—Initially, a HindIII restriction enzyme digest was performed on the human 137-kb PAC clone containing the VEGF-A gene (GenBankTM accession number AL136131 [GenBank] ). Following double gel purification (Qiagen, Valencia, CA) an 11.7-kb HindIII fragment was blunt-ended with the PCRTerminatorTM end repair kit (Lucigen, Middleton, WI). The fragment was ligated into the pSMART-LC-Kan vector using the CLONESMARTTM blunt end cloning kit (Lucigen, Middleton, WI). The resulting pSMART-LC-Kan-VEGF plasmid was digested with XhoI and the blunt end cutter EcoRV, excising an 8.2-kb fragment ranging from –6363 to +1886 kb relative to the VEGF transcription start site. The 8.2-kb fragment was then ligated into the XhoI- and SmaI-digested PGL-3-basic reporter vector (Promega, Madison, WI) creating the reporter vector pVEGF8.2-Luc. Sequencing performed by the University of New Mexico DNA Core Facility confirmed insert identity and orientation.

Dual Luciferase Assays—ARPE-19 cells were plated at 50% confluence in 12-well plates and allowed to adhere overnight. Cells in each well were transfected with a CaCl2-DNA precipitate containing: 2.5 µg of pVEGF8.2-Luc, 20 ng of pRL-CMV vector (Promega) and incubated for 4 h. Cells were then treated with 1 ml of glycerol shock solution (4x Tris-buffered saline, 15% glycerol, and 600 µM Na2HPO4) for 90 s and washed twice with TBST (142 mM NaCl, 2.7 mM KCl, 25 mM Tris, 1% Tween 20). Cells were allowed to recover for 20 h post-transfection, fed with fresh media, and treated with arsenite for the indicated times and doses. For reporter experiments with exogenous ATF4 expression, cells were infected with either Empty, ATF4 Wt, or ATF4 DN adenovirus by adding viral vectors at the time of plating. Infected cultures were then transfected with the plasmids the following day as described above. Cell lysates were obtained with passive lysis buffer (Promega) and analyzed with the Dual-luciferase® assay (Promega) using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). The firefly luciferase relative light units in each sample were normalized to the Renilla luciferase relative light units, and the sample mean ratio was divided by the control mean ratio to give fold inductions. Statistical significance of differences between triplicate samples was determined with the two-sample t test.

Western Blot Analysis—ARPE-19 or MEF cells were grown to confluence in 60 cm2 plates and treated as described in the figure legends. Following treatment, cells were washed in cold phosphate-buffered saline and lysed for 20 min on ice in lysis buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 50 mM Tris-HCl, pH 8.0) containing protease inhibitor mixture (Roche Applied Science). For samples to be analyzed with phospho-specific antibodies, phosphatase inhibitor mixture (50 mM NaF, 200 µm Na3VO4, 10 mM Na3PO4, and 100 µM EDTA) was included in the lysis buffer. Protein contents of cleared lysates were determined with a BCA protein assay kit (Pierce), and equal amounts of proteins (20 or 40 µg) were loaded into each lane and separated on a 10% SDS-PAGE gel. The protein bands were then transferred to nitrocellulose membrane (Bio-Rad) and probed with antibodies specific for ATF4 and eIF2{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated eIF2{alpha} (Cell Signaling Technology, Beverly, MA), or {beta}-actin (Sigma). Proteins were detected with ECLTM Chemiluminescence Kit (Amersham Biosciences) according to manufacture's instructions. Membranes were scanned and viewed with a MultiGenius Bioimaging System® (Syngene, Cambridge, UK).

MEF Immortalization—ATF4 heterozygous and homozygous null MEFs were obtained from Tim Townes (Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham). Cultures of 90% confluent ATF4+/– or ATF4–/– MEFs were transfected with a plasmid containing isolated SV40 early gene encoding the large T antigen alone (pD305 vector) (23) by CaCl2 co-precipitation as described above. Forty-eight h later, transfected cells were subcultured (1:10 dilution) and maintained until fully confluent (1 week). Cells were then serially diluted in 60-cm2 plates and selected for clonal growth. Approximately 2 weeks after plating, cell colonies were either isolated with cloning cylinders or harvested together with all colonies on a plate to form pooled populations. Clonal and pooled populations were expanded, and expression of large T antigen was confirmed by Western blot analysis (data not shown). Pooled populations designated as ATF4–/–/D305/1:10 (ATF4–/–) and ATF4+/–/D305/1:10 (ATF4+/–) were utilized for the present study. The presence of ATF4 protein was confirmed in the ATF4+/– population and its absence proven in ATF4–/– population by Western blotting (Fig. 4B).

DNA Binding Activity Analysis—Electrophoretic mobility shift assays were performed using Gel Shift Assay System (Promega) according to the manufacturer's protocol. The double-stranded DNA oligonucleotides containing putative ATF4-binding sites (underlined) known as amino acid response elements (AAREs) were as follows: CHOP1 AARE, 5'-TAGAGACAGGGTTTCACCATGTTGGCCAGG-3'; CHOP2 AARE in negative orientation, 5'-CCTGGGCAACATGGTGAAACACCATCTCTA-3'; DRAL AARE in negative orientation, 5'-TAGTGCACGAATGATGGAAAGGGAGGGTTG-3'; and asparagine synthetase (AsnSyn) AARE, 5'-GCGCGGAGCCGATTACATCAGCCCGGGCCT-3' (Integrated DNA Technologies, Coralville, IA). In the binding reactions, {alpha}-32P-labeled DNA probes were incubated with 10 µg of nuclear extract. Binding reactions were performed at room temperature for 20 min, and the DNA-protein complexes were separated by electrophoresis on 5% Tris borate-EDTA-PAGE gel and visualized using a STORMTM PhosphorImager and ImageQuantTM software (Amersham Biosciences). For ATF4 supershifts, 2 µg of polyclonal anti-ATF4 (CREB-2, H-290; Santa Cruz Biotechnology) or monoclonal anti-myc (9B11; Cell Signaling Technology) were incubated with the binding mixture for 10 min before loading reactions on the gel.



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  		FIG. 1.
Expression of VEGF, HO-1, and the ER stress-responsive gene GRP78 in response to various arsenite concentrations and times of arsenite exposure. A, confluent ARPE-19 cells were cultured for 4 h in medium containing 0, 5, 20, 100, and 300 µM arsenite. Northern blotting analysis with coinciding numerical values of relative VEGF mRNA inductions are shown. B, confluent ARPE-19 cells were treated with 100 µM arsenite for the indicated times. Northern blotting analysis and coinciding values for relative VEGF mRNA inductions are shown.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose Response and Time Course of VEGF, HO-1, and GRP78 mRNA Expression in ARPE-19 Cells Exposed to Arsenite—To characterize the induction of VEGF mRNA in response to arsenite, confluent ARPE-19 cells were treated with increasing doses of NaAsO2 (0, 5, 20, 100, and 300 µM) for 4 h. Total cellular RNA was isolated, and Northern blotting was performed to analyze VEGF mRNA content for each sample. (Fig. 1A). In addition, mRNA levels for HO-1 and GRP78 were examined. These genes served as indicators of oxidative and ER stress responses, respectively. In addition, ATF4 plays a role in regulation of both these genes (18, 24). The ARPE-19 cells contained detectable amounts of a single mRNA species corresponding to VEGF. The VEGF mRNA levels were normalized relative to 18 S rRNA and plotted. VEGF steady state mRNA increased maximally (5.1-fold) with 100 µM arsenite. The decrease observed with 300 µM arsenite coincided with apparent toxicity at this dosage. Cell death was not observed at lower concentrations of arsenite.

HO-1 mRNA levels were also increased by arsenite as previously described (25). HO-1 expression was much more sensitive to arsenite stress than that of VEGF. A dramatic increase in HO-1 mRNA was caused by 5 µM arsenite, representing roughly a 20-fold increase in sensitivity as compared with VEGF. Expression of GRP78/BiP mRNA was not induced by arsenite, suggesting that the unfolded protein response was not activated.

Using the dose associated with the maximal VEGF induction, 100 µM arsenite, ARPE-19 cells were treated for various times (0, 0.5, 1, 2, 4, and 8 h). This time course study revealed a maximal (4.2-fold) increase of VEGF mRNA at 4 h and a decline by 8 h. Although the sensitivity to arsenite was different for VEGF and HO-1; they exhibited similar kinetics of induction. Again, no induction of GRP78 mRNA was observed.

Activation of the VEGF Promoter by Arsenite—Further analysis of the effect of arsenite on VEGF transcription was carried out with a reporter vector, pVEGF8.2-Luc, containing 8.2 kb of the VEGF promoter region. This particular gene fragment was chosen because computer analysis of this 5' region of genomic DNA revealed it to contain four putative ATF4-binding sites, termed AARE (13). ARPE-19 cells were transfected with pVEGF8.2-Luc and pRL-CMV, allowed to recover for 20 h, treated with arsenite for the times, and doses indicated and analyzed using a dual luciferase assay. Dose-response samples were obtained after 8 h of exposure to 0, 20, 60, 100, and 140 µM arsenite. A maximal induction of 3.7-fold was achieved with 100 µM arsenite (Fig. 2A). Time course experiments were performed with exposure to 100 µM sodium arsenite for 0, 4, 6, 8, and 12 h (Fig. 2B). Arsenite induced reporter expression maximally at 8 h with an increase of 3.7-fold.



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  		FIG. 2.
Response of VEGF promoter-reporter vector to various concentrations and times of arsenite exposure. ARPE-19 cells were transiently transfected with pVEGF8.2-Luc and pRL-CMV DNA. Following transfection, cells were treated with increasing concentrations of arsenite for 8 h (A) or cells were treated with 100 µM arsenite for increasing lengths of time (B). Firefly luciferase relative light units (RLU Luc) were normalized to Renilla luciferase relative light units (RLU Ren) and data are presented as mean ± S.D., n = 3.

 
eIF2{alpha} Phosphorylation and ATF4 Protein Expression in ARPE-19 Cells Exposed to Arsenite—eIF2{alpha} phosphorylation and the subsequent up-regulation of ATF4 protein expression following arsenite treatment were expected if increased VEGF transcription was mediated by ATF4. Previous studies have reported phosphorylation of eIF2{alpha} (26) and subsequent increased in ATF4 DNA binding activity (16) upon treatment with arsenite. Therefore, the effects of 100 µM arsenite on phosphorylation of eIF2{alpha} and ATF4 protein levels in ARPE-19 cells were analyzed. Phosphorylation of eIF2{alpha} following arsenite treatment was rapid and transient (Fig. 3A). The level of phosphorylated eIF2{alpha} protein was greatly increased within 15 min and returned to basal levels within 120 min. Total eIF2{alpha} levels did not vary appreciably between the samples.

To examine the effects on ATF4 protein levels, ARPE-19 cells were treated with 100 µM arsenite for 0, 1, 2, and 4 h and analyzed by Western blotting with an antibody to ATF4. The antibody detected a prominent band of ~47 kDa, corresponding to ATF4 protein, only in samples obtained at 2 h and 4 h of arsenite treatment (Fig. 3B). The faster migrating band detected by this antibody was determined to be due to nonspecific binding, as demonstrated previously (13) and by confirming the size of endogenous ATF4 through comparison to exogenously expressed ATF4 (data not shown). {beta}-Actin was used as a loading control and did not vary markedly between samples. ATF4 protein induction coincided with VEGF mRNA induction (Fig. 1). Thus, ATF4 was available to activate VEGF transcription in a temporal manner that agrees with the time course results of VEGF mRNA induction and VEGF promoter activation.



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  		FIG. 3.
Effect of arsenite treatment on eIF2{alpha} phosphorylation and ATF4 protein expression. A, cells were treated with 100 µM arsenite for the indicated times. Western blot analysis of eIF2{alpha} phosphorylation (eIF2{alpha}-p) in ARPE-19 whole cell lysates is shown. B, cells were treated with 100 µM arsenite for the indicated times. Western blot analysis of ATF4 protein levels in ARPE-19 whole cell lysates is shown.

 
The Role of ATF4 in VEGF Expression following Arsenite Treatment—To further examine the role of ATF4 in arsenite-induced up-regulation of endogenous VEGF mRNA, an adenoviral vector system was used to express either ATF4 wild type (ATF4 Wt) or ATF4 dominant negative (ATF4 DN) proteins, as described previously (13). An empty vector (Empty) was used to control for the effects of infection and enhanced green fluorescent protein expression. Viral titers were assessed by determining the percentage of cells that exhibited enhanced green fluorescent protein fluorescence 20 h post-infection with these vectors. Viral infection of ARPE-19 cells with ATF4 Wt virus stock at a 1:320 dilution, ATF4 DN virus stock at a 1:160 dilution, and Empty virus stock at a 1:320 dilution resulted in infection rates between 90 and 100% (data not shown). ARPE-19 cells were thus infected with viruses at these dilutions of virus stocks. Twenty h after contact with the viruses, the cultures were fed with fresh media and subjected to the following: no treatment (incubated with control media), incubated with media containing 100 µM sodium arsenite, or incubated with 10 mM DL-homocysteine-containing media for 4 h. VEGF mRNA expression was then analyzed by Northern blotting (Fig. 4A). Relative VEGF mRNA levels in cells treated with empty vector were increased by arsenite (3.8-fold) and DL-homocysteine (5.5-fold). The VEGF mRNA inductions were further increased in all treatment groups when ATF4 Wt protein was overexpressed. In contrast, the expression of ATF4 DN greatly inhibited VEGF expression under all three conditions.

To confirm the effect of the ATF4 DN on VEGF expression, replicate ARPE-19 cultures were infected with ATF4 DN virus stock at a 1:120 dilution or Empty virus stock at a 1:320 dilution and 48 h later were incubated with media containing 100 µM sodium arsenite for 4 h. ATF4 DN mutant significantly reduced VEGF mRNA expression by 71% (p < 0.001, n = 3).

Heme oxygenase-1 was previously shown to be an ATF4-responsive gene via ATF4 binding on the HO-1 promoter in a complex containing the bZIP transcription factor Nrf2 (18). In ARPE-19 cells infected with empty virus, HO-1 mRNA expression was induced 31-fold by arsenite treatment (Fig. 4A). In untreated control cells, exogenous overexpression of ATF4 Wt protein increased the expression of HO-1 mRNA by 3.8-fold, whereas ATF4 DN increased HO-1 expression by 54%. ATF4 Wt overexpression had no effect on HO-1 mRNA expression in arsenite treated cells, as the level was also 31-fold that of the untreated, empty virus-infected control. Expression of ATF4 DN reduced arsenite induction of HO-1 mRNA by 29%, to 22-fold compared with the untreated, empty virus-infected control. Thus, ATF4 Wt overexpression was sufficient to induce HO-1 expression and ATF4 DN had an inhibitory effect on HO-1 induction by arsenite, but these effects were not remarkable. In contrast to arsenite, homocysteine did not induce HO-1 mRNA expression. In fact, the induction of HO-1 by exogenous ATF4 Wt expression was nearly abrogated by homocysteine treatment.

To further test the necessity of ATF4 in the induction of VEGF expression by arsenite, immortalized MEF cell strains obtained from homozygous (–/–) and heterozygous (+/–) ATF4 knock-out mice were utilized. These MEF strains were immortalized by transfection with a plasmid encoding SV40 large T antigen and selected for clonal growth. The absence of ATF4 protein expression in ATF4–/– MEF strain was demonstrated via Western blotting (Fig. 4B). Again, the antibody detected a nonspecific-immunoreactive band, however, only in lysates from ATF4+/– MEF was a band of the expected size of ATF4 protein detected. Cultures of both MEF cell strains were treated with either 100 µM arsenite or 1 µM thapsigargin for 2 h. In ATF4+/– MEF samples, the band corresponding to ATF4 greatly increased in intensity with arsenite and increased by a lesser extent with thapsigargin treatment. This band was not perceptible in ATF4–/– MEF samples, regardless of treatment.

To determine the role of ATF4 in VEGF expression, ATF4+/– and ATF4–/– MEF cell strains were treated with 100 µM arsenite or 1 µM thapsigargin for 4 h. Northern analysis of the samples demonstrated a 5.7-fold increase in VEGF mRNA levels in the arsenite-treated ATF4+/– MEF cells (Fig. 4C). The ability of ATF4–/– MEF cells to increase VEGF mRNA levels following arsenite treatment was considerably less, with only a 2-fold induction. This result confirmed the vital role of ATF4 for VEGF regulation in cells experiencing arsenite stress. The ATF4+/– MEF cells experiencing an ER stress (thapsigargin) increased VEGF expression 2.0-fold. This induction was completely abolished in the ATF4–/– MEF strain, indicating the importance of ATF4 in VEGF regulation during ER stress. Expression of the murine homologue of HO-1 was increased 90-fold in arsenite treated ATF4+/– MEF cells. ATF4–/– MEF cells exhibited a greatly diminished HO-1 response, with a 13-fold induction. Thus, ATF4 also plays a very substantial role in the arsenite induction of HO-1 in these cells. Thapsigargin induced HO-1 by 68% in ATF4+/– MEFs, and the induction was completely lost in the ATF4–/– MEFs.

Effect of Exogenous ATF4 on the VEGF Promoter in the Presence of Arsenite—The effects of exogenously expressed ATF4 on the activity of pVEGF8.2-Luc reporter vector were examined. ARPE-19 cells were infected with the adenoviral vectors containing Empty, ATF4 Wt, or ATF4 DN expression cassettes. Twenty-four h post-infection, cells were transfected with pVEGF8.2-Luc and incubated for an additional 20 h (Fig. 5A). The dual luciferase assay demonstrated that ATF4 Wt was sufficient to up-regulate the transcription of the VEGF promoter construct (6.4-fold, p = 0.005). ATF4 DN overexpression had no significant effect on VEGF transcription (1.3-fold, p = 0.38).

The combined effect of ATF4 overexpression and oxidative stress on VEGF promoter activity was then tested in ARPE-19 cells transfected as described above and treated with either control media or media containing 100 µM arsenite for 8 h (Fig. 5B). In the control cells not treated with arsenite ATF4 overexpression was again sufficient to activate the VEGF promoter by 7-fold (p = 0.005). Expression of the ATF4 DN mutant did not enhance promoter activity. These data confirmed that the VEGF promoter was responsive to ATF4 expression. Arsenite treatment enhanced expression from the VEGF promoter in cells infected with Empty vector by 2.7-fold (p = 0.002). Arsenite treatment and ATF4 overexpression acted synergistically to enhance pVEGF8.2-Luc reporter expression to 35-fold over Empty virus-infected and untreated controls. Thus, in ATF4 Wt-expressing cells the expression from the VEGF promoter was increased an additional 5-fold by arsenite treatment. Western blot analysis of ARPE-19 cells overexpressing ATF4 Wt and treated with and without arsenite demonstrated no difference in overall ATF4 expression (data not shown). Arsenite treatment did not increase expression from the VEGF promoter in ATF4 DN-expressing cells. The combination of ATF4 DN expression and arsenite insignificantly increased reporter expression (1.7-fold, p = 0.28) compared with the Empty virus control. These data indicate that the VEGF promoter is ATF4-responsive and that ATF4 and arsenite can act synergistically to activate transcription of a reporter gene linked to the VEGF promoter.



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  		FIG. 4.
Effects of inhibition of ATF4 function or lack of ATF4 expression on arsenite-induced VEGF expression. A, subconfluent cultures of ARPE-19 cells were infected with Empty, ATF4 Wt, or ATF4 DN viruses and 20 h post-infection they were treated with normal medium (Control), medium containing 100 µM arsenite (As), or medium containing 10 mM DL-homocysteine (Hcys). After 4 h of treatment, total RNA was isolated, and Northern blotting analysis was performed. B, Western blot analysis of ATF4 protein in ATF4+/– and ATF4–/– MEF cell strains treated with normal medium, 100 µM arsenite (As), or 1 µM thapsigargin (thapsi) for 2 h. C, Northern analysis and corresponding numerical values of VEGF mRNA inductions in MEF cultures. MEF cultures were treated with normal media, 100 µM arsenite (As) or 1 µM thapsigargin (thapsi) for 4 h.

 
ATF-4 Binding to AAREs Found within the VEGF Promoter Region—The activation of this particular VEGF promoter region is significant because the sequence contains putative ATF4 AARE-binding sites (also referred to as nutritional stress response elements or NSRE). Computer analysis of the 8.2-kb sequence of the VEGF promoter region contained in pVEGF8.2-Luc revealed four of these elements (Fig. 6A). Two sites identical to the AARE element within the GADD153/CHOP promoter (27) are located at positions –5668 and –4906 bp relative to the transcription start site. An AARE sequence located at –1808 bp is identical to the site found in the gene for the LIM-only protein DRAL promoter (28). Finally, an AARE at +1767 relative to the transcription start position is identical to the element in the AsnSyn promoter (referred to as NSRE-1) (29).

Once it was established that pVEGF8.2-Luc was ATF4-responsive, we tested the ability of four AARE elements that appear within the 8.2 kb fragment to bind ATF4. To obtain sources of nuclear extracts with and without ATF4 DNA binding activity, ARPE-19 cells were infected with Empty, ATF4 Wt, and ATF4 DN adenoviral vectors and incubated for 24 h. Nuclear lysates were then prepared and gel shift analysis was performed with double-stranded DNA probes that contain the four putative AARE that appear within pVEGF8.2-Luc (Fig. 6A). These AARE were assayed within the context of 30-mer double-stranded DNA oligonucleotides that retained the original surrounding sequences found in the VEGF gene. This screening assay revealed that only the +1767 AARE that resembles that from the AsnSyn gene specifically bound to ATF4-containing complex. The other AARE-containing oligonucleotides showed various degrees of nonspecific binding that was not markedly increased by ATF4 Wt expression. In contrast, the +1767 AARE exhibited no basal binding of complexes in extracts from cells treated with the Empty vector but demonstrated robust binding with extracts from ATF4 Wt-expressing cells. The ATF4 DN-containing nuclear extracts showed no +1767 AARE binding activity. The presence of ATF4 in the binding complex was demonstrated by super-shift with a monoclonal antibody targeting the myc tag of the exogenous ATF4. Excess cold oligonucleotide effectively competed for the binding complex, demonstrating specificity of +1767 AARE binding. Therefore, the screen of the putative AAREs in the sequence contexts appearing in the VEGF promoter demonstrated that only the +1767 AARE was capable of binding an ATF4-containing complex.



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  		FIG. 5.
Effects of exogenous ATF4 expression and arsenite treatment on VEGF promoter activity. ARPE-19 cells were infected with Empty, ATF4 Wt, or ATF4 DN adenoviral vectors. A, 8 h post-infection cells were transfected with the pVEGF8.2-Luc and pRL-CMV reporter vectors. Cells were harvested 24 h post-transfection, and dual luciferase assays were performed. B, ARPE-19 cells were treated with adenoviral vectors and transfected as described above; however, 20 h post-transfection, cells were treated with either normal medium (Control) or medium containing 100 µM arsenite (Arsenite) for 8 h. Cells were then harvested and dual luciferase assays were performed. For each experiment luciferase relative light units (RLU Luc) were normalized to Renilla luciferase relative light units (RLU Ren). Data are presented as mean ± S.D., n = 3. * indicates p ≤ 0.005.

 
To further evaluate the role of the +1767 AARE during stress, we next infected ARPE-19 cells with Empty, ATF4 Wt, and ATF4 DN adenoviruses for 22 h. Cells were then treated with or without 100 µM arsenite for 2 h (Fig. 6C). DNA binding was determined with the +1767 AARE-containing oligonucleotide. Arsenite treatment caused an increase in endogenous ATF4 binding in nuclear extracts of cells expressing the Empty vector. The DN mutant completely prevented ATF4 binding in extracts from cells with and without arsenite. The cells overexpressing ATF4 Wt protein displayed high DNA binding activity, and arsenite treatment caused a further increase in this activity. Thus, the synergism observed with ATF4 Wt and arsenite treatment (Fig. 5B) coincided with increased +1767 AARE binding activity. ATF4 was present in the DNA-binding complex as demonstrated by supershift with the anti-myc antibody and complex disruption by a polyclonal ATF4 antibody. These data demonstrated that the putative +1767 AARE was capable of binding ATF4 and that this binding was increased with arsenite treatment.

Effect of Oxidative Stress on ATF4-dependent VEGF Expression—It was shown previously that NAC and glutathione were capable of inhibiting VEGF mRNA induction in response to arsenite treatment (9). We tested the ability of several antioxidants to inhibit VEGF mRNA inductions by arsenite treatment of ARPE-19 cells (Fig. 7). Cells were pretreated for 30 min with either: no antioxidant, 10 mM NAC, 1 mM trolox (soluble vitamin E analogue), 1000 units/ml catalase, or 10 mM ascorbic acid (vitamin C). Following preincubation with the antioxidants, the cells received either no treatment (controls) or were treated with 100 µM arsenite for 4 h. As demonstrated previously (9), VEGF induction by arsenite was completely abolished by NAC (Fig. 7, A and B). However, VEGF induction was only partially inhibited by trolox and not affected by catalase or vitamin C. The effects of these antioxidants on HO-1 induction by arsenite was also examined. In contrast to VEGF, HO-1 induction was only slightly inhibited by NAC but was markedly inhibited by trolox and vitamin C. Catalase did not affect the induction of HO-1.

To determine whether inhibition of VEGF induction by NAC was due to its ability to inhibit eIF2{alpha} phosphorylation and ATF4 up-regulation, the effect of NAC on these responses following arsenite treatment was tested. ARPE-19 cells were pretreated with no antioxidant (control) or 10 mM NAC for 30 min, and then 0 or 100 µM arsenite was added, and cells were incubated for 2 h. Western blot analysis showed a nearly complete inhibition of arsenite-induced ATF4 up-regulation by NAC (Fig. 7C). NAC alone had no effect on basal ATF4 levels. To test whether eIF2{alpha} phosphorylation was inhibited by NAC, cells were pretreated with NAC or no antioxidant and treated with 100 µM arsenite for 0, 5, 15, 30, and 60 min. Western blot analysis of total cell lysates indicated that eIF2{alpha} phosphorylation was effectively inhibited by NAC.

We tested whether the inhibition of ATF4-related signaling by NAC was due to reactivity between its thiol group and arsenite in the media. To examine this possibility, equimolar concentrations of arsenite and NAC were combined and incubated at room temperature for time periods up to 24 h, and thiol concentrations were determined using 5,5'-dithiobis-2-nitrobenzoic acid. The thiol reactivity of NAC was not diminished by arsenite, even after 24 h of incubation (data not shown). This demonstration suggested that NAC does not interact directly with arsenite in the media and thus does not simply spare the cells from arsenite exposure.



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  		FIG. 6.
Analysis of ATF4 binding to the four AARE found in the VEGF promoter. A, a schematic of the four putative AARE found in the VEGF promoter. B, ARPE-19 cells were infected with Empty, ATF4 Wt, and ATF4 DN adenoviral vectors for 24 h. Nuclear extracts were prepared and DNA binding activities of the extracts were assayed with 30-mer double-stranded DNA oligonucleotides corresponding to each putative AARE in the sequence context found in the VEGF gene. C, ARPE-19 cells were infected with Empty, ATF4 Wt, and ATF4 DN adenoviral vectors for 22 h. Cultures were then exposed to control media (AS–) or 100 µM arsenite (AS+) for 2 h. Nuclear extracts were prepared and gel shift analysis was performed with the +1767 AARE-containing oligonucleotide.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study includes the following novel findings. 1) Arsenite induced the expression of VEGF mRNA by a retinal pigment epithelial cell line in a time- and dose-dependent manner. 2) Arsenite induced transcriptional up-regulation of a VEGF promoter construct. 3) Arsenite-induced eIF2{alpha} phosphorylation was followed by an increase in ATF4 protein level. 4) Induction of VEGF expression by arsenite was diminished by expression of a dominant negative ATF4 mutant and in ATF4–/– MEF cells. 5) The VEGF promoter is ATF4-responsive and arsenite cooperated with exogenous ATF4 overexpression to superinduce the promoter. 6) The AsnSyn AARE was capable of binding ATF4 in the context found in the VEGF promoter. 7) NAC treatment abolished arsenite induced eIF2{alpha} phosphorylation, ATF4 protein expression, and up-regulation of VEGF mRNA.

The ability of exogenous ATF4 delivered by adenoviral infection to activate the promoter in the absence of stress suggests that the VEGF gene is not only responsive to ATF4 but that ATF4 is sufficient to up-regulate VEGF expression. The synergism seen between ATF4 and arsenite suggests either the involvement of an arsenite-responsive transcription factor, such as an ATF4 binding partner, or activation of overexpressed ATF4 in arsenite-treated cells. For example, phosphorylation of ATF4 by RSK2 (p90 ribosomal S6 kinase) has recently been implicated in ATF4 activation (30). ATF4 is also a NIPK (neuronal cell death-inducible putative kinase (also referred to as "tribbles 3" or "TRB3")) substrate, and phosphorylation by NIPK enhances transactivation (31). Although phosphorylation may be important, Western analysis did not demonstrated a slower migrating ATF4 protein band consistent with phosphorylation of ATF4 in arsenite-treated cells (data not shown). Electrophoretic mobility shift assay using the AARE found at +1767 bp confirmed that the DNA binding activity of an ATF4-containing complex was increased with arsenite treatment, but the mechanism by which this occurred remains undetermined.



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  		FIG. 7.
Effects of antioxidants on ATF4-mediated VEGF expression in arsenite treated ARPE-19 cells. A, Northern blot analysis of VEGF and HO-1 relative mRNA inductions in cells pretreated with antioxidants and treated with arsenite (As). ARPE-19 cells were pretreated with either no antioxidant medium (Control and As) or medium containing 10 mM NAC, 1 mM trolox, 1000 units/ml catalase, or 1 mM vitamin C (Vit C) for 30 min. Following preincubation with the antioxidants, cells were treated with either no treatment (Control) or with 100 µM arsenite for 4 h. B, corresponding numerical values of mRNA inductions obtained by Northern analysis of cells treated with normal media (Control), 100 µM arsenite (As), or 100 µM arsenite following pretreatment for 30 min with 10 mM NAC. Data are presented as mean ± S.D., n = 3. C, Western blot analysis of ATF4 protein levels in ARPE-19 cells treated with arsenite and NAC. Cells were pretreated for 30 min with either no antioxidant media (Control and As) or with 10 mM NAC. Pretreated cells were then treated with no treatment (Control and NAC) or with 100 µM arsenite for 2 h. D, Western blot analysis for eIF2{alpha} phosphorylation (eIF2{alpha}-P) in ARPE-19 cells treated with arsenite and NAC. After no pretreatment or pretreatment for 30 min with 10 mM NAC, the cells were treated with 100 µM arsenite for the times indicated.

 
Four AARE matching those found in other ATF4-responsive genes were identified in the 8.2-kb VEGF promoter region used in this study. When tested in the sequence contexts found in the VEGF gene, and only the AsnSyn AARE at +1767 bp demonstrated binding by an ATF4-containing complex. The necessity of this element for transcriptional activation of VEGF in response to oxidative stress and other stresses leading to ATF4 expression needs to be tested.

Antioxidants varied in their abilities to prevent VEGF and HO-1 expression following arsenite treatment. Neither the VEGF response nor the HO-1 response was inhibited by catalase treatment. Thus, either H2O2 does not play a significant role in the VEGF and HO-1 response or external catalase treatment was simply ineffective at detoxifying intracellular H2O2. Vitamin C did not affect VEGF induction but did dramatically decrease the HO-1 response. Trolox, a soluble vitamin E analogue, had a modest effect on VEGF up-regulation but almost completely abolished the HO-1 response. The trolox effect on VEGF suggests some involvement of arsenite-generated ROS in VEGF regulation. The most dramatic VEGF inhibition was seen with NAC, yet NAC had very little effect on HO-1 mRNA levels.

Phosphorylation of eIF2{alpha} and induction of ATF4 protein level preceded the induction of VEGF mRNA level. The presence of ATF4 following arsenite stress suggests that ARPE-19 cells activate signaling cascades for protection from arsenite-induced oxidative stress. ATF4 is thought to be protective against oxidative stress through its ability to support glutathione biosynthesis (17). NAC was able to inhibit eIF2{alpha} phosphorylation and ATF4 protein expression as well as inhibit VEGF mRNA induction. The ability of NAC to inhibit ATF4 protein increase and eIF2{alpha} phosphorylation further implicates this signaling pathway in the VEGF response to arsenite-induced redox stress.

The sensory kinase involved in eIF2{alpha} phosphorylation following arsenite treatment is of interest because of its possible role as a sensor of oxidative stress. PERK is activated by accumulation of unfolded proteins in the ER and by ER Ca2+ perturbation (32). PKR is activated in the presence of double-stranded RNA and by interferon (33), and its phosphorylation is compounded by arsenite (34). HRI regulates globin synthesis to match heme availability in developing reticulocytes (35) and may regulate eIF2{alpha} phosphorylation in response to certain oxidative stresses. Arsenite treatment activates HRI in reticulocytes, and HRI null erythroid cells do not induce eIF2{alpha} phosphorylation following arsenite treatment (36). A recent comparison of MEF knock-out cells demonstrated that HRI was the only known eIF2{alpha} kinase required for the translational inhibition in response to arsenite (37).

In summary, the results of the present study have shown that ATF4 is a positive regulator of VEGF transcription in arsenite-treated ARPE-19 cells. These results suggest a mechanism by which ATF4 might regulate VEGF expression in retinal cells exposed to similar oxidative stresses. Because numerous stresses cause activation of eIF2{alpha} kinases leading to increased ATF4 expression, this mechanism could contribute to VEGF expression in response to a variety of non-hypoxic stresses. Also, because ATF4 is an important regulator of both cellular protection against oxidative stress and VEGF expression, further examination of the role of this transcription factor in AMD and DR is warranted.


   FOOTNOTES
 
* This work was supported by Grant EY13695 from the NEI/National Institutes of Health (NIH) (to D. L. V.), Research Fellowship EY014535 from the NEI/NIH (to C. N. R.), and Grant DAMD17-03-1-0588 from the Department of Defense Breast Cancer Research Program (to S. F. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, MSC08-4670, 1-University of New Mexico School of Medicine, Albuquerque, NM 87131-0001. Tel.: 505-272-4138; Fax: 505-272-3836; E-mail: sabcouwer{at}salud.unm.edu.

1 The abbreviations used are: AMD, age-related macular degeneration; DR, diabetic retinopathy; VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor; ER, endoplasmic reticulum; ATF, activating transcription factor; HO, heme oxygenase; NAC, N-acetylcysteine; MEF, mouse embryo fibroblast; Wt, wild type; DN, dominant negative; AARE, amino acid response element; AsnSyn, asparagine synthetase. Back


   ACKNOWLEDGMENTS
 
We thank Stephanie Tran for her help in transfecting and maintaining the ATF4+/– and ATF4–/– MEF clones during the immortalization process.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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