Evidence for a Role of p38 Kinase in Hypoxia-inducible Factor 1-independent Induction of Vascular Endothelial Growth Factor Expression by Sodium Arsenite*

Monique C. A. DuyndamDagger, Saskia T. M. Hulscher, Elsken van der Wall, Herbert M. Pinedo, and Epie Boven

From the Department of Medical Oncology, Vrije Universiteit Medical Center, Amsterdam 1081 HV, The Netherlands

Received for publication, June 25, 2002, and in revised form, December 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1alpha (HIF-1alpha ) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1alpha and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1alpha protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1alpha -/- mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vasular Endothelial Growth Factor (VEGF)1 plays a key role in tumor angiogenesis. VEGF occurs in at least six isoforms of 121, 145, 165, 183, 189, and 206 amino acids, which are generated from a single gene by alternative splicing (1). We have reported that VEGF165 overexpression stimulates angiogenesis in human ovarian cancer xenografts (2).

Elevated expression of VEGF in tumor cells can be the result of environmental factors, such as hypoxia, or mutations in oncogenes or tumor suppressor genes that regulate growth factor signal transduction pathways (3-9). Many stimuli, including hypoxia, growth factors, hormones, and oxidative stressors, can increase VEGF expression in tumor cells in vitro (6, 10-14). In all mentioned cases, increased VEGF expression can be caused in part by increased VEGF gene transcription mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) (4-7, 11, 13-15). HIF-1 binds to a hypoxia-responsive element (HRE) located within the VEGF promoter (11).

HIF-1 is composed of two subunits, HIF-1alpha and HIF-1beta . The activity of HIF-1 is regulated mainly by the expression and activity of the HIF-1alpha subunit. Although HIF-1beta protein is rather stable and readily detected in the nucleus of most normoxic cells, HIF-1alpha protein is often hardly detectable because of rapid degradation by the ubiquitin-proteasome system (16-18). Hypoxia increases the level of HIF-1alpha protein by inhibiting its ubiquitination and degradation (19). Accumulation of HIF-1alpha protein can also be observed in stimulated normoxic cells (5, 8, 9, 13-15). HIF-1alpha is subsequently translocated to the nucleus, where it can dimerize with HIF-1beta to form the HIF-1 complex (20). To be fully active, HIF-1alpha requires interaction(s) with coactivators or/and transcription factor(s) (20-23). The activated protein-1 transcription factor family member c-Jun interacts with HIF-1alpha and is suggested to cooperate with HIF-1 in the induction of VEGF expression by hypoxia (23).

The stabilization and transcriptional activation of the HIF-1alpha protein involve changes in its phosphorylation state. Activation of the lipid kinase phosphatidylinositol 3-kinase (PI3K), and/or its downstream target the protein-serine/threonine kinase Akt, can result in the phosphorylation and stabilization of HIF-1alpha under hypoxic and normoxic conditions (8, 24). In addition, inhibition of PI3K activity has been shown to reduce the transactivation function of HIF-1alpha in hypoxic cells (25). So far, there is no evidence that PI3K and Akt can phosphorylate HIF-1alpha directly. Other pathways that regulate HIF-1alpha phosphorylation involve members of the mitogen-activated protein kinase (MAPK) family (26). In hypoxic and in stimulated normoxic cells, p44/p42 MAPK (extracellular signal-regulated kinase (ERK)-1 and ERK-2) and p38 MAPK enhance the transactivation function of HIF-1alpha (24, 25, 27). This may occur through direct phosphorylation because these kinases have been shown to phosphorylate HIF-1alpha in vitro (28, 29). In some cases, p44/p42 MAPK and p38 can also influence HIF-1alpha protein induction (8, 30). The stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs) do not phosphorylate HIF-1alpha in vitro, but may indirectly regulate HIF-1-mediated transcription of VEGF under hypoxia through phosphorylating the transcription factor c-Jun (18, 23).

The stabilization and transcriptional activation of HIF-1alpha may also involve alterations in its redox state. Evidence is provide that changes in the levels of reactive oxygen species (ROS) may play a role in HIF-1alpha protein induction and HIF-1 transactivation (14, 30-32). Increased levels of ROS are suggested to mediate PI3K activation under hypoxia as well as under normoxia (32, 33). The levels of ROS may also directly or indirectly influence the redox status of cysteine residues in the transactivation domains of HIF-1alpha , which can affect interactions with transcriptional coactivators (21, 22).

In a previous study on the role of oxidative stress in the regulation of VEGF, we showed that sodium arsenite (NaAsO2) induces HIF-1alpha protein and VEGF mRNA and protein levels in the human ovarian cancer cell lines OVCAR-3 and H134 (34). Arsenite induces oxidative stress by binding to thiol groups of cellular proteins and by increasing the production of ROS. Because the effects of sodium arsenite on HIF-1alpha protein and VEGF expression are independent of increased ROS production, we hypothesized that they may be mediated through binding of arsenite to thiol (SH) groups of the HIF-1alpha protein itself or of components of signal transduction pathways involved in HIF-1 or VEGF regulation (34).

Our findings with sodium arsenite may be of clinical relevance. Several cytotoxic agents in cancer treatment are susceptible of interacting with thiol groups of cellular proteins. Moreover, arsenic trioxide (As2O3), another trivalent arsenic compound, has potential as an anticancer agent (35). At low dosages (1-10 µM), arsenic trioxide has a significant cytotoxic effect on human ovarian cancer cell lines and is suggested to be a useful agent for the treatment of ovarian cancer (36). Therefore, we now compared the potency of arsenic trioxide with that of sodium arsenite to induce HIF-1alpha protein and VEGF mRNA and protein levels in the OVCAR-3 human ovarian cancer cell line. We also investigated the role of the PI3K/Akt pathway and of MAPK family members in sodium arsenite-induced HIF-1alpha protein accumulation and VEGF expression. Furthermore, we examined whether up-regulation of VEGF mRNA expression by sodium arsenite was mediated by HIF-1.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Chemicals-- Sodium arsenite, arsenic trioxide, wortmannin, glutathione (GSH), N-acetylcysteine (NAc), and buthionine-sulfoximine (BSO) were purchased from Sigma. PD98059, SB203580 and SB202190 were purchased from Calbiochem.

Cell Culture and Cell Treatment-- OVCAR-3 human ovarian cancer cells and Jnk1+/- Jnk2-/-, Jnk1-/-Jnk2-/-, HIF-1alpha +/+, and HIF-1alpha -/- mouse fibroblasts were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Sanbio, Uden, The Netherlands), 50 units/ml penicillin (ICN Biochemicals, Zoetermeer, The Netherlands), and 50 µg/ml streptomycin (ICN Biochemicals). Cells were routinely cultured in 95% air and 5% CO2 at 37 °C. Hypoxic conditions were performed by incubation of cells in a tightly sealed chamber maintained at 1% oxygen, 94% N2, and 5% CO2 at 37 °C. Immortalized Jnk1+ - Jnk2-/- and Jnk1-/- Jnk2-/- cell lines were kindly provided by Dr. E. F. Wagner (Research Institute of Molecular Pathology, Vienna, Austria). These cell lines have been established independently from individual primary mouse embryo fibroblasts that were each isolated from a single mouse embryo following the 3T3 protocol (37, 38). Immortalized HIF-1alpha +/+ and HIF-1alpha -/- cell lines were kindly provided by Dr. G. L. Semenza (Johns Hopkins University School of Medicine, Baltimore, MD). Both cell lines were established by transfection of primary mouse embryo fibroblast cultures with an expression vector encoding simian virus 40 T-antigen as described elsewhere (39).

For treatment of cells with sodium arsenite or arsenic trioxide, cells were seeded in culture dishes in medium and grown overnight. Thereafter, sodium arsenite or arsenic trioxide was added to the conditioned medium, and cells were incubated further for the time periods as indicated in each experiment. Pretreatment of cells with wortmannin, PD98059, SB203580, SB202190, GSH, and NAc was performed for 1 h before the addition of sodium arsenite; pretreatment with BSO was performed for 16 h.

Antibodies-- Rabbit polyclonal antisera directed against phospho-Akt-1 (Ser473), phospho-p44/p42 MAPK (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-c-Jun (Ser73), p44/p42 MAPK, SAPK/JNK, p38 MAPK, and a horseradish peroxidase-coupled anti-rabbit antiserum were purchased from New England Biolabs. Mouse monoclonal antisera to HIF-1alpha were purchased from Novus Biologicals/AbCam (Cambridge, U. K.) and BD Transduction Laboratories (Alphen a/d Rijn, The Netherlands). The sheep polyclonal antiserum directed against Akt-1 was from Upstate Biotechnology Inc. Rabbit polyclonal antisera directed against c-Jun (H-79) and Raf-1 (C12) were purchased from Santa Cruz Biotechnology. The mouse monoclonal antiserum against beta -actin (C4) was purchased from Roche Molecular Biochemicals. The polyclonal human antiserum against topoisomerase I was purchased from TopoGEN (Columbus, OH). The mouse monoclonal antiserum against human p53 (DO-7) and the horseradish peroxidase-coupled anti-mouse serum were purchased from DAKO (Glostrup, Denmark). The horseradish peroxidase-coupled anti-sheep serum was purchased from Calbiochem.

Preparation of Cell Extracts for Western Blot Analysis and ELISA-- Cells were washed once with ice-cold phosphate-buffered saline and lysed by scraping with a rubber policeman in 250 µl of radioimmune precipitation assay buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, and 1% sodium deoxycholate) for Western blotting or in 450 µl of E1A buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, and 0.1% Nonidet P-40) for ELISA. Both lysis buffers were supplemented with 50 mM NaF, 1 mM Na3VO4, 1.0 mM phenylmethylsulfonyl fluoride, 0.5 mM trypsin inhibitor, and 0.5 µg/ml leupeptin. After a 15-min incubation period on ice, the extracts were clarified by centrifugation at 14,000 rpm for 15 min at 4 °C and stored at -70 °C. Isolation of nuclear and cytoplasmic protein fractions from OVCAR-3 cells in the subcellular fractionation experiment was performed as described previously (40). Protein concentrations were determined by the Coomassie Plus Protein assay (Pierce).

Western Blotting-- Equal amounts of protein cell extracts were resolved in SDS-polyacrylamide gels and transferred electrophoretically onto a polyvinylidene difluoride membrane (Immobilon). Membranes were blocked for 1 h in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.025% Tween 20) and 5% milk and incubated overnight with antiserum directed against phospho-Akt-1(1:3,000), phospho-p44/p42 MAPK (1:1,000), phospho-SAPK/JNK (1:1,000), phospho-p38 (1:1,000), phospho-c-Jun (1:1,000), HIF-1alpha (1:500), beta -actin (1:2,000), Raf-1 (1:500), topoisomerase I (1:5,000) or p53 (1:1,000). After washing with TBST, the membranes were incubated for 1 h with horseradish peroxidase-linked anti-rabbit or anti-mouse antiserum in TBST and 5% milk. The membranes were washed again with TBST, and proteins were visualized by enhanced chemiluminescence. To detect both nonphosphorylated and phosphorylated forms of Akt-1, c-Jun, and the MAPK family members, membranes were stripped for 15 min in strip buffer (10 mM Tris, pH 8.0, 2% SDS, 10 mM beta -mercaptoethanol) at 42 °C and washed with TBST. Subsequently, the blots were blocked again and incubated for 2 h with Akt-1, p44/p42 MAPK, SAPK/JNK, or p38-specific antiserum (1:1,000 dilution) or with a c-Jun-specific antiserum (1:500 dilution) in TBST and 5% milk. The incubations with horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-sheep antisera and the detection of the proteins were performed as described above.

ELISA-- Equal numbers of cells were plated on 9.6-mm culture dishes and grown overnight. The conditioned medium of all dishes was collected, pooled, and 450 µl was sampled (time-point (T) = 0 medium sample). Cells of one dish were washed and lysed in 450 µl of lysis buffer as described above (T = 0 lysate sample). The conditioned medium was divided into 5 volumes. Sodium arsenite was added to 1 volume at a concentration of 100 µM, and arsenic trioxide was added to 3 volumes at concentrations of 100, 50, and 10 µM. Subsequently, conditioned medium with or without sodium arsenite or arsenic trioxide was again added to culture dishes with cells. Thus, at T = 0 the amount of VEGF protein in the medium was the same in each culture dish. Cells were incubated further for the time periods indicated in each experiment. Thereafter, 450 µl of conditioned medium was sampled, and cells were lysed. VEGF concentrations in nondiluted media samples and lysates were determined in duplicate by ELISA using the reagents and the protocol supplied with the Quantikine Human VEGF Immunoassay kit (R&D Systems). Differences in VEGF concentrations in medium and lysates of nontreated versus sodium arsenite- or arsenic trioxide-treated cells were evaluated using Student's t test for two groups. p values <0.05 were considered to be significant.

RNase Protection Assay-- Generation of human gamma -actin and VEGF165 antisense probes has been described elsewhere (34). pcDNA3 vectors (Invitrogen) containing a fragment of murine VEGF164 or gamma -actin cDNA were used as templates for the synthesis of murine VEGF164 and gamma -actin antisense probes. The templates were generated as follows. Total RNA from Jnk1+/- Jnk2-/- mouse fibroblasts was reversed transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Murine VEGF164 cDNA (nucleotides 454-648) was amplified with the forward primer 5'-ATAACAAGCTTAGCACAGCAGATGTGAATGC-3' and the reversed primer 5'-GCAACCTCGAGCTTGTCACATCTGCAAGTAC-3'. Murine gamma -actin cDNA (nucleotides 630-780) was amplified with the forward primer 5'-ATAACAAGCTTGCTATGTTGCCCTGGATTTTGAG-3' and the reversed primer 5'-GCAACCTCGAGGGA AGGAAGGCTGGAAGAGT-3'. Amplification of VEGF164 and gamma -actin cDNA fragments was performed for 36 cycles at 94 °C, 56 °C, and 72 °C. In addition to VEGF164 or gamma -actin sequences (underlined), the forward and reverse primers contain restriction sites (bold) for HindIII and XhoI, respectively. The 216-nucleotide VEGF164 and 172-nucleotide gamma -actin amplified fragments were extracted with phenol/chloroform, precipitated, dissolved in water, and digested with HindIII and XhoI. The digestion mixtures were subjected to gel electrophoresis, and the VEGF164 and gamma -actin cDNA fragments were isolated from the agarose gel by use of the QIAquick gel extraction kit (Qiagen). The isolated VEGF164 and gamma -actin cDNA fragments were cloned into the HindIII and XhoI restriction sites of pcDNA3, and the sequence of the VEGF164 and gamma -actin cDNA fragments was verified by sequencing. pcDNA3VEGF164 and pcDNA3gamma -actin were linearized with HindIII, and 228-nucleotide VEGF164 and 185-nucleotide gamma -actin antisense probes were generated with SP6 polymerase.

The RNase protection assay was carried out as described (34). Hybridization of total RNA to a 136-nucleotide human gamma -actin antisense probe gives rise to a protected fragment of 130 nucleotide, whereas hybridization to the 185-nucleotide murine gamma -actin antisense probe is expected to result in the protection of a fragment of 151 nucleotides. It should be noted that hybridization of total RNA to the 301-nucleotide human VEGF165 antisense probe and the 228-nucleotide murine VEGF164 can give rise to fragments of different sizes because of protection by VEGF mRNAs of different isoforms (1, 34, 41, 42). Protection by human VEGF165 mRNA and murine VEGF164 mRNA is most efficient, giving rise to protected fragments of 252 and 195 nucleotides, respectively. Therefore, effects on VEGF mRNA levels were monitored by assessing the mRNA levels of human VEGF165 and murine VEGF164.

Plasmid Constructs-- The pGL3 promoter vector was purchased from Promega. This vector contains the SV40 basal promoter upstream of luciferase coding sequences and the SV40 late polyadenylation signal. 5xHREpGL3 contains five copies of a HIF-1 consensus sequence of the human VEGF promoter and was cloned from the reporter plasmid 5HRE/hCMVmp (43). 5HRE/hCMVmp was digested with KpnI and BglII, and the KpnI-BglII fragment was ligated into the KpnI-BglII-digested pGL3-promoter vector. 5xjun2pGL3 contains five copies of the jun2 element of the human c-jun promoter (44) and was constructed by ligation of the PvuII-BglII fragment of the 5xjun2-tata-luciferase reporter construct (45) in the SmaI-BglII-digested pGL3 promoter vector.

Transient Transfection and Luciferase Assay-- OVCAR-3 cells were seeded on six-well culture plates and grown overnight in medium. Transfection of OVCAR-3 cells with luciferase reporter constructs was performed in duplicate wells by the calcium-phosphate precipitation method (46). After a 6-h incubation period with the precipitate, the cells were washed and refed with culture medium. 40 h later, cells were left untreated or exposed to sodium arsenite or hypoxia for 6 h. Cells were washed with phosphate-buffered saline and lysed in 250 µl of lysis reagent (25 mM Tris phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM CDTA, 10% glycerol, 1% Triton X-100). Protein concentrations were determined by the Coomassie Plus Protein assay and beta -galactosidase activity was assessed as described by Sambrook et al. (47). Luciferase activity was determined by mixing 30 µl of cell extract with 100 µl of luciferase assay reagent (Promega) and subsequent measurement of relative light units for 10 s in a luminometer (Lumat LB 9507, EG&G Berthold, Bad Wildbad, Germany). The mean relative luciferase activity was calculated as the mean luciferase activity (relative light units)/beta -galactosidase (A420/µg of protein/min) of duplicate extracts.

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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High Concentrations of Arsenic Compounds Induce HIF-1alpha Protein and VEGF Expression in OVCAR-3 Cells-- We compared the effects of different concentrations of arsenic trioxide with those of sodium arsenite on HIF-1alpha protein, VEGF mRNA, and VEGF protein expression in OVCAR-3 cells (Fig. 1). As can be seen in Fig. 1A, high concentrations of arsenic trioxide (100 and 50 µM) could indeed induce HIF-1alpha protein after 4 and 8 h in OVCAR-3 cells, but to a much lesser extent than an equimolar concentration of sodium arsenite (100 µM). We also tested the effect of 8- and 24-h exposure periods to 10 µM arsenic trioxide. This treatment did not induce detectable changes in HIF-1alpha protein expression in OVCAR-3 cells.


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Fig. 1.   Effect of sodium arsenite and trivalent arsenic on the levels of HIF-1alpha protein, VEGF165 mRNA, and VEGF165 protein in OVCAR-3 cells. OVCAR-3 cells were exposed to 100 µM sodium arsenite or to 100, 50, or 10 µM arsenic trioxide for the indicated time periods. After collection of conditioned medium, cells were lysed, and protein and total RNA were extracted. A, HIF-1alpha protein levels were assessed by subjecting 100 µg of protein to SDS-gel electrophoresis (7.5% gel) followed by Western blotting with a HIF-1alpha -directed antiserum. An unidentified protein that is nonspecifically recognized by the HIF-1alpha -directed antiserum or by the secondary horseradish peroxidase-linked antiserum is indicated as NS. B, VEGF165 mRNA levels were assessed by the RNase protection assay. Total RNA was hybridized to human VEGF165 and gamma -actin antisense probes. t-RNA (lane T) was hybridized as a negative control. The 252- and 130-nucleotide fragments protected by the mRNAs of VEGF165 and gamma -actin and the full-length probes are indicated. The histogram indicates the relative VEGF165 mRNA expression in sodium arsenite or arsenic trioxide-treated OVCAR-3 cells in which nontreated controls (T = 0) were set at 100%. C, VEGF165 protein concentrations in conditioned media (left panel) and lysates (right panel) of sodium arsenite or arsenic trioxide-treated OVCAR-3 cells were determined by ELISA. As a control, VEGF protein concentrations were also measured in conditioned media and lysates of nontreated cells. The histograms represent the mean ± S.D. of duplicate samples in a representative experiment. Significant differences in mean VEGF concentrations in medium and lysates of nontreated versus sodium arsenite or arsenic trioxide-treated cells after individual incubation periods are indicated by an asterisk (p < 0.05). Results of sodium arsenite and arsenic trioxide as shown in A, B, and C were assessed in parallel in one experiment that was representative of three independent ones giving comparable results.

The effects of arsenic trioxide on VEGF mRNA expression were assessed by the RNase protection assay (Fig. 1B). As determined by measurement of the signal intensities of the 252-nucleotide VEGF165- and 130-nucleotide gamma -actin-protected fragments, VEGF165 mRNA levels in OVCAR-3 cells were increased 3- and 5-fold after 4 and 8 h of exposure to 100 µM arsenic trioxide, respectively. A 2- and 3-fold increase in VEGF165 mRNA expression was observed after 4 and 8 h of exposure to 50 µM arsenic trioxide. An 8-h exposure period to low concentrations of arsenic trioxide (10 µM) did not influence the level of VEGF165 mRNA, whereas a weak elevation (1.5-fold) was observed after 24 h of exposure. In agreement with the effects on HIF-1alpha protein, arsenic trioxide at 100 µM was a less potent inducer of VEGF165 mRNA than sodium arsenite. 100 µM sodium arsenite increased the level of VEGF165 mRNA up to 6- and 7-fold after 4 and 8 h of exposure.

We next assessed whether induction of VEGF mRNA levels by arsenic trioxide resulted in an increased production of VEGF protein. OVCAR-3 cells were grown overnight, and arsenic trioxide and sodium arsenite were added to the conditioned medium. VEGF concentrations in the conditioned medium or in cell lysates of treated and nontreated cells were measured by ELISA. As can be seen in Fig. 1C, VEGF protein levels in the conditioned medium were increased significantly after 8 h of exposure to 100 and 50 µM of arsenic trioxide compared with the levels in the medium of control cells (p < 0.05). In the lysates, increased production of VEGF was evident after 4 h of incubation and was more pronounced after 8 h (p < 0.05). As expected, a higher increase in VEGF protein concentrations in conditioned medium and lysate was observed after treatment with 100 µM sodium arsenite. Note that a weak elevation of VEGF protein levels was detected in conditioned medium and lysate of cells that were exposed to 10 µM arsenic trioxide for 24 h. These results clearly show that arsenic trioxide induces HIF-1alpha protein accumulation and VEGF expression in OVCAR-3 cells, albeit less potently than sodium arsenite. In addition, these results indicate that long exposure to a low, clinically relevant concentration of arsenic trioxide can weakly increase VEGF165 mRNA and protein expression in the absence of a detectable change in the level of HIF-1alpha protein expression.

Induction of HIF-1alpha Protein and VEGF Expression by Sodium Arsenite Is Not Mediated through PI3K-- Because it has been suggested that sodium arsenite can activate PI3K in some cell types, we investigated the role of this pathway in sodium arsenite-induced HIF-1alpha protein accumulation and VEGF expression in OVCAR-3 cells (48). PI3K-mediated activation of Akt-1, the most frequently studied isoform of Akt, involves phosphorylation on its amino acid residues Ser473 and Thr308. We first monitored the phosphorylation of Akt-1 upon sodium arsenite treatment in OVCAR-3 cells by Western blotting with an antiserum recognizing Ser473-phosphorylated Akt-1. As can be seen in Fig. 2A, OVCAR-3 cells showed a relatively high basal level of phospho-Akt-1. After 15 min of exposure to 100 µM sodium arsenite, Akt-1 phosphorylation on Ser473 was increased, which was sustained until at least 4 h of exposure. After 8 h, the levels of phospho-Akt-1 were decreased to basal. The same results were obtained with an antiserum directed against Thr308 (data not shown). The levels of total Akt-1 were unchanged upon sodium arsenite treatment for up to 8 h. We examined further the involvement of PI3K in sodium arsenite-induced Akt-1 phosphorylation by assessing the effects of the PI3K inhibitor wortmannin. Fig. 2B shows that pretreatment with 100 nM wortmannin completely blocked induction of Akt-1 phosphorylation by sodium arsenite. Wortmannin alone did not significantly affect the basal level of phospho-Akt-1. Again, the total levels of Akt-1 protein remained constant. These results suggest that sodium arsenite indeed activates the PI3K/Akt-1 pathway in OVCAR-3 cells.


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Fig. 2.   Effect of wortmannin on sodium arsenite-induced Akt-1 phosphorylation, HIF-1alpha protein accumulation, and VEGF expression in OVCAR-3 cells. OVCAR-3 cells were exposed to 100 µM sodium arsenite in the absence or presence of 100 or 500 nM wortmannin or to 100 or 500 nM wortmannin alone. Wortmannin was added 1 h before the addition of sodium arsenite. After the indicated periods of exposure to sodium arsenite, cells were lysed, and protein and total RNA were extracted. A and B, the phosphorylation of Akt-1 was monitored by subjecting 100 µg of protein to SDS-gel electrophoresis (8% gel) followed by Western blotting with an antiserum specific to Ser(P)473. The blots were stripped and reprobed with a total Akt-1 antiserum. C, HIF-1alpha protein levels were examined by subjecting 100 µg of protein to SDS-gel electrophoresis (7.5% gel) followed by Western blotting with a HIF-1alpha -directed antiserum. D, VEGF165 mRNA levels were analyzed by RNase protection as in B.

We next investigated the effect of wortmannin on sodium arsenite-induced HIF-1alpha protein accumulation by analyzing HIF-1alpha protein levels in the extracts of Fig. 2A by Western blot. In sharp contrast to the phosphorylation of Akt-1, induction of HIF-1alpha protein by sodium arsenite was completely unaffected by 100 nM wortmannin (Fig. 2C). Pretreatment with wortmannin concentrations as high as 500 nM only weakly inhibited HIF-1alpha protein accumulation. Analysis of total RNA by the RNase protection assay in parallel samples revealed that neither sodium arsenite-induced VEGF165 mRNA levels nor the basal VEGF165 mRNA levels in OVCAR-3 cells were influenced by pretreatment with 100 and 500 nM concentrations of wortmannin (Fig. 2D). These results strongly suggest that induction of HIF-1alpha protein and VEGF expression by sodium arsenite is independent of the PI3K/Akt-1 pathway in OVCAR-3 cells.

Sodium Arsenite activates p44/p42 MAPK, SAPK/JNK, and p38 with Different Kinetics in OVCAR-3 Cells-- Depending on the cell type, sodium arsenite can activate different MAPK family members (49), which may contribute to HIF-1alpha protein accumulation and VEGF expression. Therefore, we first monitored the activity of p44/p42 MAPK, SAPK/JNK, and p38 upon sodium arsenite treatment in OVCAR-3 cells by detection of their phosphorylated, activated forms on Western blots.

As can be seen in Fig. 3, nonstimulated OVCAR-3 cells showed strong phosphorylation of p42/p44 MAPK. This is consistent with a previous study reporting a high basal activity of p44/p42 MAPK in these cells (50). A weak increase in the phosphorylation of p44/p42 MAPK was observed as early as 5 min after the addition of 100 µM sodium arsenite and remained detectable until at least 1 h of exposure. The phosphorylation of both kinases returned to basal levels after 2 h and was even reduced to lower levels after 4 and 8 h. Sodium arsenite also induced the activity of JNK1, JNK2, and p38, albeit with different kinetics. A strong increase in the phosphorylation of JNK1, JNK2, and p38 was observed after 1 h of sodium arsenite treatment, and increased phosphorylation of all three kinases was prolonged until at least 8 h of treatment. Changes in phosphorylation of p42/p44 MAPK, JNK1, JNK2, and p38 were not accompanied by alterations in the levels of these proteins. These data indicate that sodium arsenite activates p44/p42 MAPK, JNK1, JNK2, as well as p38 with different kinetics in OVCAR-3 cells. Activation of p44/p42 MAPK occurred rapidly but was only moderate and transient. In contrast, activation of JNK1, JNK2, and p38 was slower but more potent and sustained.


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Fig. 3.   Effect of sodium arsenite on the phosphorylation of MAPK family members in OVCAR-3 cells. OVCAR-3 cells were exposed to 100 µM sodium arsenite for the indicated time periods. 25 µg of protein was subjected to SDS-gel electrophoresis (10% gel) and Western blotting. Phosphorylation of p44/p42 MAPK, JNK1/2 (p46, p54), and p38 was detected using phospho-antisera. The blots were stripped and reprobed with antisera recognizing both nonphosphorylated and phosphorylated forms of p44/p42 MAPK, JNK1/2 (p46, p54), and p38.

Sodium Arsenite-induced HIF-1alpha Protein Accumulation and VEGF Expression Are Attenuated by Inhibitors of p38 MAPK Activity-- To examine further the involvement of p44/p42 MAPK and p38 in the induction of HIF-1alpha protein and VEGF expression by sodium arsenite, we analyzed the effect of the kinase inhibitors PD98059, SB202190, and SB203580 in OVCAR-3 cells. PD98059 is a specific inhibitor of MAPK/ERK kinase (MEK)-1 and MEK-2, which phosphorylate and activate p44/p42 MAPK. SB202190 and SB203580 directly inhibit the activity of p38 by binding to its ATP binding domain.

Fig. 4, A-C, shows that induction of HIF-1alpha protein and VEGF165 mRNA expression after 4 and 8 h of exposure to 100 µM sodium arsenite was slightly, but not significantly, reduced by pretreatment with high concentrations of PD98059 (50 µM). The basal level of HIF-1alpha protein and VEGF165 mRNA was also hardly affected by this agent. PD98058 was functional in inhibiting p44/p42 MAPK activity at the concentrations used, as demonstrated by the reduced level of phospho-p44/p42 MAPK in the presence and absence of sodium arsenite (Fig. 4D). In fact, phospho-p44/p42 MAPK levels were even below basal and hardly detectable after 4 and 8 h of sodium arsenite and PD98059. These data indicate that p44/p42 MAPK does not regulate induction of HIF-1alpha protein and VEGF expression in OVCAR-3 cells.


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Fig. 4.   Effect of kinase inhibitors on sodium arsenite-induced HIF-1alpha protein and VEGF mRNA levels in OVCAR-3 cells. OVCAR-3 cells were exposed to 100 µM sodium arsenite in the absence or presence of the kinase inhibitors PD98059 (50 µM), SB202190 (20 µM), and SB203580 (20 and 5 µM) or to the individual kinase inhibitors alone. The kinase inhibitors were added 1 h before the addition of sodium arsenite. After the indicated periods of exposure to sodium arsenite, cells were lysed, and protein and RNA were extracted. A, HIF-1alpha protein levels were assessed as in Fig. 2C. B, VEGF165 mRNA levels were determined as in Fig. 1B. C, the relative VEGF165 mRNA expression was determined by signal intensity measurements of VEGF165- and gamma -actin-protected fragments on autoradiographs and subsequent normalization of VEGF165 values against gamma -actin internal control values. The values in the bar graph are the means ± S.D. of the relative VEGF165 mRNA expression of three independent experiments in which nontreated controls were set at 100%. Significant differences between the relative VEGF165 mRNA expression after incubation with sodium arsenite and that of control cells are indicated by an asterisk (p < 0.05). D, phosphorylation of p44/p42 MAPK was examined as described in Fig. 3. E, phosphorylation of c-Jun was monitored by sub- jecting 100 µg of protein to SDS-gel electrophoresis (10% gel) followed by Western blotting with an antiserum specific to Ser(P)73. The blot was stripped and reprobed with an antiserum recognizing both nonphosphorylated and phosphorylated forms of c-Jun.

Pretreatment with the p38 inhibitors SB202190 and SB203580 could potently inhibit induction of HIF-1alpha protein and VEGF mRNA expression by sodium arsenite (Fig. 4, A-C). As assessed at 4 and 8 h after the addition of 100 µM sodium arsenite, both effects were almost completely attenuated by a 20 µM concentration of each inhibitor. Significant inhibition of HIF-1alpha protein accumulation and VEGF mRNA expression was also observed by pretreatment with 5 µM SB202190 (data not shown) and SB203580 (Fig. 4, A-C). Treatment with the two inhibitors alone did not significantly influence the basal level of HIF-1alpha protein and VEGF165 mRNA in OVCAR-3.

In some cell types, high concentrations (20-100 µM) of SB202190 or/and SB203580 can inhibit the activity of JNKs (51). Activated JNKs are known to enhance the activity of the transcription factor c-Jun through the phosphorylation of two serine residues at positions 63 and 73 (26). To examine whether JNK activity was influenced by SB203580 in sodium arsenite-treated OVCAR-3 cells, we analyzed the phosphorylation of c-Jun on Ser73 and the total level of c-Jun by Western blotting. Fig. 4E shows that phosphorylated c-Jun proteins were not detectable in nonstimulated OVCAR-3 cells. After 2 h of sodium arsenite treatment, phosphorylation of c-Jun protein was clearly observed and was increased further until at least 8 h of exposure. Consistent with findings that phosphorylation on Ser63 and Ser73 decreases the electrophoretic mobility of the c-Jun protein (52), slower migrating forms of c-Jun were detectable after sodium arsenite treatment. The total level of c-Jun protein was also increased by sodium arsenite. Pretreatment with 20 µM SB203580 did not prevent the phosphorylation of c-Jun on Ser73 in OVCAR-3 cells upon exposure to sodium arsenite (Fig. 4E). Hyperphosphorylated, retarded c-Jun protein band(s) were clearly detectable. The observed decrease in the level of phospho-c-Jun protein is more likely the result of an inhibitory effect of SB203580 on sodium arsenite-induced c-Jun protein expression instead of inhibition of c-Jun phosphorylation. Pretreatment with 5 µM SB203580 did also not affect c-Jun phosphorylation and had only a minor effect on c-Jun protein levels. These results support that attenuation of sodium arsenite-induced HIF-1alpha protein accumulation and VEGF mRNA expression by SB203580 is specific and exclude the possibility that this is caused by a toxic effect of this inhibitor on OVCAR-3 cells. In addition, these data indicate that SB203580 does not influence JNK activity in sodium arsenite-treated OVCAR-3 cells at the concentrations used. Therefore, our results with SB203580 and SB202190 strongly suggest that induction of HIF-1alpha protein and VEGF expression by sodium arsenite is mediated by p38 in OVCAR-3 cells.

Activation of p38 MAPK and Induction of HIF-1alpha Protein by Sodium Arsenite Are Co-modulated by the Level of Intracellular GSH-- We have shown previously that the level of intracellular GSH is critical for induction of HIF-1alpha protein and VEGF expression by sodium arsenite in OVCAR-3 cells (34). Pretreatment with agents that elevate intracellular GSH levels, such as GSH and NAc (a precursor of GSH), attenuate sodium arsenite-induced HIF-1alpha accumulation and VEGF expression, whereas depletion of intracellular GSH by pretreatment with BSO potentiates both effects (34). The latter agent causes GSH depletion by inhibiting gamma -glutamylcysteine synthetase, an enzyme involved in the synthesis of GSH (53).

In light of our findings with the p38 inhibitors that p38 may be an upstream regulator of HIF-1alpha protein and VEGF expression, we assessed whether activation of p38 by sodium arsenite was also modulated by exposure to GSH, NAc, and BSO prior to the addition of sodium arsenite in OVCAR-3 cells. As a control, the level of the HIF-1alpha protein was analyzed in parallel in the same extracts. Fig. 5A shows that pretreatment with 10 and 20 mM GSH and NAc almost completely attenuated sodium arsenite-induced phosphorylation of p38 in OVCAR-3 cells. Differences in the signal of the phospho-p38 band were not caused by changes in the total level of p38 because these remained almost constant under all circumstances. The basal level of p38 phosphorylation appeared not to be influenced by pretreatment with 20 mM concentrations of the antioxidants alone. In agreement with our previous findings (34), GSH and NAc pretreatment also attenuated HIF-1alpha protein induction, whereas a minor effect was observed on the basal level of HIF-1alpha protein.


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Fig. 5.   Effect of GSH, NAc, and BSO pretreatment on induction of p38 phosphorylation by sodium arsenite in OVCAR-3 cells. A, OVCAR-3 cells were exposed to 100 µM sodium arsenite in the absence or presence of 10 or 20 mM GSH or 10 or 20 mM NAc. GSH and NAc were added 1 h before the addition of sodium arsenite. In addition, cells were exposed to 20 mM GSH and 20 mM NAc alone. After an 8-h exposure to sodium arsenite, whole cell extracts were prepared. HIF-1alpha protein levels and phosphorylation of p38 were assessed by subjecting 100 µg of protein to SDS-gel electrophoresis (7.5% gel) followed by Western blotting with a HIF-1-directed antiserum or with a phospho-p38 antiserum. For assessing total protein levels of p38, the blot was stripped and reprobed with an antiserum recognizing both nonphosphorylated and phosphorylated forms of p38. B, OVCAR-3 cells were exposed to 30 µM sodium arsenite in the absence or presence of 500 µM BSO. BSO was added 16 h before the addition of sodium arsenite. Cells were also exposed to 500 µM BSO alone. After 4, 6, and 8 h of exposure to sodium arsenite, whole cell extracts were prepared. HIF-1alpha protein levels and the phosphorylation of p38 were assessed as in A.

To examine the effect of GSH depletion on p38 phosphorylation, OVCAR-3 cells were pretreated with 500 µM BSO for 16 h before the addition of a lower, suboptimal concentration of sodium arsenite (30 µM). Because sodium arsenite reduces intracellular GSH levels by itself, the effect of BSO is more pronounced when OVCAR-3 cells are exposed to a suboptimal concentration of this agent. Fig. 5B shows that pretreatment with BSO clearly potentiated the phosphorylation of p38 after 4, 6, and 8 h of exposure to sodium arsenite, without affecting the total levels of this protein. Pretreatment with 500 µM BSO alone for a period of 24 h hardly affected the basal levels of p38 phosphorylation. It should be mentioned that potentiation of p38 activity in the presence of BSO was already observed after 4 h, whereas potentiation of HIF-1alpha protein accumulation was not detected until 6 h of exposure to sodium arsenite. Thus, the p38 response in GSH-depleted OVCAR-3 cells upon sodium arsenite treatment precedes the response of HIF-1alpha . This result is consistent with the role of p38 as an upstream regulator of HIF-1alpha protein induction by sodium arsenite. In addition, the above data further support that regulation of HIF-1alpha accumulation and VEGF expression by intracellular GSH in sodium arsenite-treated OVCAR-3 cells involves modulation of p38 activity.

JNK1 and JNK2 Are Not Essential for Induction of HIF-1alpha Protein and VEGF164 mRNA Expression by Sodium Arsenite in Mouse Fibroblast Cell Lines-- Unfortunately, examination of the role of JNKs in sodium arsenite-treated OVCAR-3 cells is complicated by the lack of pharmacological agents that specifically inhibit their activity. Therefore, we have examined whether sodium arsenite can affect HIF-1alpha protein and VEGF mRNA levels in two mouse fibroblast cell lines that were deficient of Jnk1 and Jnk2. We also analyzed a mouse fibroblast cell line in which one of the four Jnk allelles, a Jnk1 allele, was wild type. The lack of JNK activity in the two Jnk1-/- Jnk2-/- fibroblast cell lines was confirmed by Western blotting demonstrating the absence of Ser73-phosphorylated c-Jun proteins in extracts of sodium arsenite-treated cells (Fig. 6A). In contrast, phosphorylation of c-Jun was potently induced in sodium arsenite-treated Jnk1+/- Jnk2-/- cells (Fig. 6A). As can be seen in Fig. 6B, a clear induction of HIF-1alpha protein was observed in the two Jnk1-/- Jnk2-/- cell lines after 4 and 8 h of exposure to 100 µM sodium arsenite. The increase in the level of HIF-1alpha protein upon sodium arsenite treatment was more pronounced than that observed in Jnk1+/- Jnk2-/- fibroblasts. In all three cell lines, the levels of HIF-1alpha protein were highest after 4 h of treatment with sodium arsenite.


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Fig. 6.   Effect of sodium arsenite on HIF-1alpha protein and/or VEGF164 mRNA levels in Jnk1- Jnk2-deficient mouse fibroblast cell lines. Jnk1+/- Jnk2-/- and Jnk1-/- Jnk2-/- mouse fibroblast cell lines were treated with 100 µM sodium arsenite for the indicated time periods before cells were lysed and protein and total RNA were extracted. A, c-Jun phosphorylation and HIF-1alpha protein levels were assessed as in Figs. 4E and 2C, respectively. B, VEGF164 mRNA levels were examined by the RNase protection assay. Total RNA was hybridized to murine VEGF164 and gamma -actin antisense probes. t-RNA (lane T) was hybridized as a negative control. The 195-nucleotide fragment protected by the mRNAs of VEGF164 is indicated. Hybridization of gamma -actin mRNAs to the gamma -actin antisense probe resulted in the protection of three different fragments, including the expected fragment of 151 nucleotides (indicated by an arrow, see text below). The full-length VEGF164 and gamma -actin antisense probes are shown in the two leftmost lanes. The histogram indicates the relative VEGF165 mRNA expression in sodium arsenite-treated cells in which nontreated control cells (T = 0) were set at 100%. Quantification was performed by signal intensity measurements of VEGF164 and gamma -actin (151-nucleotide)-protected fragments on the autoradiograph of the RNase protection experiment shown and subsequent standardization of VEGF164 values against gamma -actin internal control values. The experiments in A and B are representative of three independent ones with similar results.

The effect of sodium arsenite on the levels of VEGF164 mRNA in these cell lines was determined in parallel by the RNase protection assay. As can be seen in Fig. 6B, a VEGF164-protected fragment with the expected size of 195 nucleotides and a low signal intensity was detected in nontreated Jnk1-/- Jnk2-/- as well as in Jnk1+/- Jnk2-/- fibroblasts. Protection of a 151-nucleotide gamma -actin fragment was also observed in all three cell lines. The VEGF164- and gamma -actin-protected fragments were not detected after hybridization of the antisense probes to control tRNA. In addition, two other protected fragments were visualized which were smaller in size. The protection of these fragments was specific and, although not easily explained, may also be caused by hybridization by gamma -actin mRNAs. The levels of VEGF164 mRNA in the two Jnk1-/- Jnk2-/- cell lines were increased 3-4-fold after 4 h of treatment with 100 µM sodium arsenite. In Jnk1+/- Jnk2-/- fibroblasts, induction of VEGF mRNA expression by sodium arsenite was also detectable, but was less pronounced (2-fold). Elevated levels of VEGF164 mRNA were still observed after 8 h of exposure to arsenite in all three cell lines. In summary, our results in mouse fibroblast cell lines demonstrate that induction of HIF-1alpha protein and VEGF mRNA levels by sodium arsenite is not restricted to human ovarian cancer cells. More importantly, the data suggest that JNK1 and JNK2 are not essential for these effects.

HIF-1alpha Protein Is Translocated to the Nucleus in Sodium Arsenite-treated OVCAR-3 Cells-- So far, it is unclear whether induction of HIF-1alpha protein by sodium arsenite leads to the formation of functional HIF-1. Because the translocation of HIF-1alpha to the nucleus is an important regulatory event in the activation of HIF-1, we first examined the subcellular localization of HIF-1alpha in nontreated and sodium arsenite-treated OVCAR-3 cells. Because hypoxia has been shown to induce efficient nuclear translocation of HIF-1alpha in several cell types (20), we assessed the subcellular distribution of HIF-1alpha in hypoxic OVCAR-3 cells as a control. Cytoplasmic and nuclear protein fractions were extracted from nontreated OVCAR-3 cells and from cells exposed to 100 µM sodium arsenite or hypoxia (1% O2) for 6 h, and identical amounts of protein extract were subjected to SDS-PAGE and Western blotting. In addition, whole cell extracts were prepared and analyzed in parallel. Fig. 7 shows that a low level of HIF-1alpha protein expression was detected in whole cell extracts of nontreated OVCAR-3 cells and that the level of HIF-1alpha protein was potently induced upon exposure to sodium arsenite as well as to hypoxia (34). A low level of HIF-1alpha protein was observed in the nuclear fraction but not in the cytoplasmic fraction of nontreated OVCAR-3 cells. Much higher levels of HIF-1alpha were observed in the nuclear fractions as well as in the cytoplasmic fractions of OVCAR-3 cells that were exposed to sodium arsenite and hypoxia. As a control for the fractionation procedure, we also analyzed the levels of proteins with a known subcellular localization (Fig. 7). As assessed in whole cell extracts, the levels of the cytoplasmic proteins Raf-1 and beta -actin the nuclear proteins topoisomerase I and p53 (mutant conformation) were unaffected by treatment with sodium arsenite and hypoxia. Raf-1 and beta -actin were indeed present primarily in the cytoplasmic fractions, whereas topoisomerase I and p53 were detected mainly in the nuclear fractions. These control tests confirmed that the fractionation procedure was valid. Altogether, these data strongly indicate that HIF-1alpha is transported to the nucleus in nontreated OVCAR-3 cells and that HIF-1alpha protein accumulates in the nucleus and in the cytoplasm of OVCAR-3 cells upon exposure to sodium arsenite and hypoxia.


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Fig. 7.   Determination of the localization of HIF-1alpha in nontreated and hypoxia- or sodium arsenite-treated OVCAR-3 cells by biochemical subcellular fractionation. OVCAR-3 cells were either not treated or exposed to 1% oxygen (hypoxia) or 100 µM sodium arsenite under normoxia for 6 h. Cytoplasmic and nuclear fractions as well as whole cell extracts were prepared, and 100 µg of protein was subjected to SDS-gel electrophoresis (7.5% gel) and Western blotting with antisera directed against HIF-1alpha , beta -actin, Raf-1, topoisomerase I, and p53.

Induction of HIF-1alpha Protein by Sodium Arsenite May Not Lead to the Formation of Functional HIF-1 in OVCAR-3 Cells-- To examine further the functional activity of HIF-1alpha upon sodium arsenite treatment, OVCAR-3 cells were transiently transfected with a 5xHREpGL3 luciferase reporter gene construct containing five HRE binding sites from the human VEGF promoter in front of the SV40 minimal promoter. The empty pGL3 promoter construct, containing only the SV40 minimal promoter in front of the luciferase gene, was transfected as a negative control. We also transfected a 5xjun2pGL3 reporter construct, which contains five copies of the jun2 element from the c-jun promoter in front of the SV40 minimal promoter. The jun2 element binds c-Jun/ATF-2 heterodimers and is suggested to mediate induction of c-jun gene expression by agents that induce different types of cellular stress, including sodium arsenite (54, 55). As mentioned earlier, c-Jun is a substrate of JNKs, whereas ATF-2 is phosphorylated and activated by JNKs as well as by p38 (26). Because c-jun mRNA levels were elevated in sodium arsenite-treated OVCAR-3 cells (data not shown), we transfected the 5xjun2pGL3 reporter construct as a positive control.

As can be seen in Fig. 8, the 5xHREpGL3 construct was not induced by 50 and 100 µM sodium arsenite as measured after 6 h of exposure. To exclude the possibility that the 5xHREpGL3 construct was not functional, transfected OVCAR-3 cells were exposed for the same period of 6 h to hypoxia in parallel. Note that HIF-1alpha protein expression was strongly induced after 6 h of exposure to hypoxia as well as after 6 h of exposure to 100 µM sodium arsenite (Fig. 7). In agreement with findings in other cell types, the expression of the 5xHREpGL3 reporter gene was significantly greater (7-fold) than that of the parental pGL3 construct in hypoxic OVCAR-3 cells. This suggests that the 5xHREpGL3 construct is functional in mediating transcriptional responses through HIF-1. The 5xjun2pGL3 construct was induced ~2-fold after treatment with 50 and 100 µM sodium arsenite. This activation is actually 3-fold when normalized for the activity of the parental pGL3 construct under the same conditions. Although the kinetics of activation of c-Jun/ATF-2 and HIF-1 upon sodium arsenite treatment may differ, our findings with the 5xjun2pGL3 construct suggest that the assay conditions used allow detection of increased expression of pGL3 reporter constructs in arsenite-treated OVCAR-3. Altogether, these data indicate that induction of HIF-1alpha protein by sodium arsenite may not lead to the formation of functional HIF-1 in OVCAR-3 cells.


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Fig. 8.   Induction of HIF-1alpha protein by sodium arsenite does not result in HIF-1-mediated transcriptional activation. OVCAR-3 cells were transiently transfected in duplicate wells of six-well culture plates with 0.5 µg of the indicated pGL3-luciferase reporter constructs together with 0.25 µg of pCMVLacZ and 2.0 µg of pUC19 carrier DNA (per well). pCMVLacZ was cotransfected to correct for a variable transfection efficiency. 40 h after transfection, cells were left untreated or exposed to 50 or 100 µM sodium arsenite or 1% oxygen (hypoxia) for an additional period of 6 h. The relative luciferase activity in the histogram represents the mean luciferase (RUL)/beta -galactosidase ratio/µg of total protein/min ± S.D. of duplicate extracts. Three independent experiments were performed with similar outcome. The results of one of them are shown.

HIF-1alpha Is Not Essential for Induction of VEGF Expression by Sodium Arsenite in Mouse Fibroblasts-- To examine further the role of HIF-1 in sodium arsenite-induced VEGF mRNA expression, we have studied the effect of this agent on VEGF mRNA expression in a wild type and a HIF-1alpha -deficient mouse fibroblast cell line (39, 56). As shown in the RNase protection assay in Fig. 9, unstimulated HIF-1alpha +/+ as well as HIF-1alpha -/- fibroblasts express a very low level of VEGF164 mRNA. After 4 h of exposure to sodium arsenite, VEGF164 mRNA levels were elevated ~2.5-fold in HIF-1alpha +/+ cells. In HIF-1alpha -/- cells, the increase in VEGF164 mRNA at this time point was even higher, 3.5-fold. In both cell types, the gamma -actin mRNA levels were not altered upon sodium arsenite treatment. These findings in mouse fibroblasts strongly suggest that HIF-1(alpha ) does not play a role in the up-regulation of VEGF mRNA expression by sodium arsenite and support our suggestive data in OVCAR-3 cells that induction of HIF-1alpha protein by sodium arsenite does not result in the generation of functional HIF-1.


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Fig. 9.   Effect of sodium arsenite on VEGF164 mRNA levels in HIF-1alpha +/+ and HIF-1alpha -/- mouse fibroblast cell lines. HIF-1alpha +/+ and HIF-1alpha -/- mouse fibroblast cell lines were treated with 100 µM sodium arsenite for the indicated time periods before cells were lysed and total RNA was extracted. VEGF164 mRNA levels were examined by the RNase protection assay as in Fig. 6B.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have demonstrated previously that sodium arsenite induces HIF-1alpha protein as well as VEGF mRNA and protein expression in the human ovarian cancer cell lines OVCAR-3 and H134 (34). Here, we show that 50 and 100 µM arsenic trioxide, an experimental anticancer agent, exerts the same effects in OVCAR-3 cells. Although treatment of OVCAR-3 cells with 10 µM arsenic trioxide for a relatively long period of exposure (24 h) did not influence HIF-1alpha protein levels, a weak elevation was observed in the expression of VEGF165 mRNA (1.5-fold). The levels of VEGF protein in conditioned medium and in cell lysate were increased 3- and 2-fold, respectively. These findings suggest that at low, clinically achievable concentrations of arsenic trioxide, ovarian cancer cells may respond with up-regulation of VEGF protein.

The finding that arsenic trioxide induces HIF-1alpha protein and VEGF expression was not unexpected because different arsenic compounds containing trivalent arsenic have been shown to exert similar biological effects (49). At least part of these effect are believed to be caused by the ability of trivalent arsenic to bind to SH groups of important cellular (signaling) proteins. Both sodium arsenite and arsenic trioxide can activate PI3K and/or the MAPK family members p44/p42 MAPK, SAPKs/JNKs, and p38 (48, 49). We demonstrated that these kinases were indeed activated by sodium arsenite in OVCAR-3 cells. Our experiments with the PI3K inhibitor wortmannin and the p44/p42 MAPK inhibitor PD98059 strongly suggest that sodium arsenite-induced HIF-1alpha protein and VEGF mRNA expression are independent of PI3K/Akt-1 and p44/p42 MAPK. Interestingly, phenylarsine oxide, another trivalent arsenic compound, can also induce HIF-1alpha protein through a PI3K/Akt-1-independent mechanism (57).

Recently, it was suggested that the transcription factor c-Jun cooperates with HIF-1 in the activation of VEGF and that JNKs mediate this activation by phosphorylating c-Jun on Ser63 and Ser73 (23). We showed induction of HIF-1alpha protein and VEGF165 mRNA in immortalized Jnk1+/- Jnk2-/- and Jnk1-/- Jnk2-/- mouse fibroblast cell lines by sodium arsenite, which did not involve c-Jun phosphorylation on Ser63 and Ser73. Thus, our data provide strong evidence that JNK activity is not essential for the effects of sodium arsenite on HIF-1alpha and VEGF.

The p38 MAPK inhibitors SB202190 and SB203580 completely abolished induction of HIF-1alpha protein and VEGF165 mRNA expression by sodium arsenite in OVCAR-3 cells. A role of p38 as an upstream activator in the effects of sodium arsenite on HIF-1alpha and VEGF was supported by additional observations. First, activation of p38 was observed after 1 h of exposure to sodium arsenite and clearly preceded the induction of HIF-1alpha protein and VEGF165 mRNA levels (observed after 4 and 2 h, respectively) (34). Second, we found p38 kinase activation as well as HIF-1alpha protein and VEGF165 mRNA expression to be co-modulated by the level of intracellular GSH (34). Elevation of intracellular GSH by pretreatment with GSH or NAc (a precursor for GSH) inhibited the effects of sodium arsenite on HIF-1alpha protein and VEGF165 mRNA expression in OVCAR-3 cells, whereas depletion of intracellular GSH by BSO showed the reverse (34). We demonstrated here that pretreatment with GSH, NAc, and BSO influenced the effects of sodium arsenite on p38 activity in the same manner. Thus, p38 may act as a mediator in the modulation of HIF-1alpha accumulation and VEGF expression by intracellular GSH. Third, we have transient cotransfected OVCAR-3 cells with an expression vectors containing either a puromycin resistance gene or a gene encoding an unphosphorylatable dominant-negative mutant of p38 (DNp38). As assessed by real time quantitative PCR, induction of VEGF165 mRNA by sodium arsenite after 3 days of selection with puromycin was partly inhibited (20-40%) upon cotransfection of DNp38 in three independent experiments. The partial inhibition is likely the result of suboptimal selection of transfected cells with puromycin. Upon stable cotransfection, 19 puromycin-resistant OVCAR-3 clones did not show detectable expression of DNp38, indicating that overexpression of DNp38 may inhibit OVCAR-3 cell growth. For further studies, the generation of OVCAR-3 cells with inducible expression of DNp38 may be required.

Although a role for p38 in the regulation of HIF-1alpha protein levels under normoxia has been suggested in some studies, this kinase has mainly been implicated in the regulation of HIF-1alpha transactivation (25, 29). The mechanism by which p38 exerts its effect on HIF-1alpha , however, is still unclear. We have provided evidence that sodium arsenite induces the level of HIF-1alpha protein by inhibiting its degradation (34). The degradation of HIF-1alpha is controlled by a small domain of 200 amino acids (amino acids 401-603), called the oxygen-dependent degradation domain (17). Interestingly, this oxygen-dependent degradation domain contains three serine residues (at positions 581, 589, and 594) which may be putative targets for direct phosphorylation by p38. The oxygen-dependent degradation domain mediates the interaction of HIF-1alpha with the tumor suppressor protein von Hipple Lindau (pVHL) under normoxic conditions (58). pVHL is part of a multiprotein complex possessing associated E3 ubiquitin-ligase activity and is thought to target the degradation of HIF-1alpha (58, 59). Phosphorylation of Ser581, Ser589, or Ser594 by p38 may disrupt or prevent the formation of the HIF-1alpha ·pVHL complex. It is, however, questionable whether HIF-1alpha is a direct target for p38 in sodium arsenite-treated OVCAR-3 cells. Activation of p38 already occurs after 1 h of sodium arsenite treatment, whereas HIF-1alpha protein accumulation is not detectable until 4 h (34). This suggests that p38 may regulate the HIF-1alpha protein indirectly, through phosphorylation of other factors. A possible candidate is FK506 binding protein-rapamycin-associated protein (FRAP), an effector of Akt, which may be activated by sodium arsenite in a p38-dependent manner (60). FRAP has been suggested to mediate hypoxia- and growth factor-induced HIF-1alpha protein expression (6).

The finding that up-regulation of HIF-1alpha protein as well as VEGF mRNA by arsenite is blocked by p38 kinase inhibitors would support that induction of VEGF expression is mediated by HIF-1. Elevation of VEGF mRNA expression in sodium arsenite-treated OVCAR-3 cells, however, was detected at earlier time points (after 2 h) than HIF-1alpha protein stabilization (after 4 h) (34). Moreover, exposure of OVCAR-3 cells to sodium arsenite did not result in transcriptional activation of a 5xHRE-dependent reporter gene in a transient transfection assay. Furthermore, treatment of OVCAR-3 with low concentrations of arsenic trioxide also up-regulated VEGF mRNA expression in the absence of a detectable change in the level of HIF-1alpha protein. Taken together, these data indicate that up-regulation of VEGF expression by arsenite and arsenic trioxide is mediated by other factors than HIF-1. This is supported by experiments in HIF-1alpha +/+ and HIF-1alpha -/- mouse fibroblast cell lines, demonstrating that elevation of VEGF mRNA levels upon sodium arsenite treatment is even slightly higher in HIF-1alpha -/- cells than in HIF-1alpha +/+ cells.

It is unclear why HIF-1 is not functional in sodium arsenite-treated OVCAR-3 cells. Our results from subcellular fractionation experiments showing that HIF-1alpha protein is clearly detectable in nuclear cell fractions of sodium arsenite-treated OVCAR-3 cells suggest that HIF-1alpha protein is properly translocated to the nucleus. It remains to be explored whether HIF-1alpha can dimerize to HIF-1beta and whether this complex is able to bind to DNA and to transcriptional coactivators. Several studies suggest that the DNA binding activity of HIF-1 and its interactions with transcriptional coactivators is dependent on its redox state (21, 22, 61). Addition of sulfhydryl-reactive agents to extracts of hypoxic cells can inhibit HIF-1 DNA binding (61). These agents can also inhibit the binding of HIF-1alpha to coactivator(s) (21). Possibly, arsenic agents inhibit the binding of HIF-1 to DNA or to coactivators through direct interaction with cysteine residues. The former hypothesis is supported by the finding that HIF-1alpha protein induction by phenylarsine oxide does not result in the formation of an active HIF-1 DNA-binding complex (57). In agreement with our results on sodium arsenite, this compound did also not provoke HRE-dependent reporter gene activation (57).

Recently, evidence was provided that VEGF mRNA can be stabilized in response to p38 activation under normoxic conditions (62). Experiments with the transcription inhibitor actinomycin D suggested, however, that induction of VEGF mRNA levels by sodium arsenite is at the transcriptional level (34). Besides the HRE, the VEGF promoter contains binding sites for several other transcription factors that can mediate transcriptional activation of VEGF in a HIF-1-independent manner under normoxic conditions (63, 64). As a first attempt to identify the transcription factors that mediate sodium arsenite-induced VEGF expression in OVCAR-3 cells, we performed transient transfection assays with luciferase reporter constructs containing the full-length VEGF promoter (-2274/+379) (11). Although the expression of this reporter construct was inducible by hypoxia in OVCAR-3 cells, we have never observed any activation of this reporter by sodium arsenite. Additional experiments are required to establish whether the effect of sodium arsenite on VEGF expression is the result of transcriptional activation or of mRNA stabilization. Further investigation of the mechanism that mediates sodium arsenite-induced VEGF expression and of the exact role of p38 in this effect will provide important knowledge on the regulation of VEGF expression under conditions of oxidative stress.

    ACKNOWLEDGEMENTS

We are grateful to Dr. E. F. Wagner (Vienna, Austria) for providing the Jnk1+/- Jnk2-/- and Jnk1-/- Jnk2-/- mouse fibroblast cell lines. We also acknowledge the generosity of Dr. G. L. Semenza (Baltimore, MD) for providing the HIF-1alpha +/+ and HIF-1alpha -/- mouse fibroblast cell lines. We thank Dr. W. P. J. Leenders (Nijmegen, The Netherlands), Dr. T. Shibata (Kyoto, Japan), Dr. H. van Dam (Leiden, The Netherlands), and Dr. E. Nishida (Kyoto, Japan) for the use of the BSVEGF165, the 5HRE/hCMV, the 5xjun-tata-luciferase reporter construct and the Sralpha DNp38 construct, respectively. We thank D. Fontijn for technical assistance.

    FOOTNOTES

* This work was supported by the Walter Bruckerhoff Stiftung.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.

Dagger To whom correspondence should be addressed: Dept. of Medical Oncology, De Boelelaan 1117, Amsterdam 1081 HV, The Netherlands. Tel.: 31-20-444-8327; Fax: 31-20-444-4355; E-mail: mca.duyndam. oncol{at}med.vu.nl.

Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M206320200

    ABBREVIATIONS

The abbreviations used are: VEGF, vascular endothelial growth factor; BSO, buthionine-sulfoximine; CDTA, trans-1,2-diaminocyclohexane-N,N,N'N'-tetraacetic acid; CMV, cytomegalovirus; DN, dominant-negative; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; HIF-1, hypoxia-inducible factor 1; HRE, hypoxia-responsive element; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; NAc, N-acetylcysteine; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; SAPK, stress-activated protein kinases; VHL, von Hippel Lindau; T, time point.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Robinson, C. J., and Stringer, S. E. (2001) J. Cell Sci. 114, 853-865[Abstract/Free Full Text]
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