AMP-activated protein kinase activity is required for vanadate-induced hypoxia-inducible factor 1{alpha} expression in DU145 cells

Jin-Taek Hwang1, Minyoung Lee1, Seung-Nam Jung1, Hye-Jeong Lee2, Insug Kang1, Sung-Soo Kim1 and Joohun Ha1,3

1 Department of Biochemistry and Molecular Biology, Medical Research Center for Bioreaction to Reactive Oxygen Species, Kyung Hee University College of Medicine, Seoul 130-701, Korea and 2 Department of Pharmacology, Medical Research Center for Cancer Molecular Therapy, Dong-A University, Busan, 602-103, Korea

3 To whom correspondence should be addressed Email: hajh{at}khu.ac.kr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hypoxia-inducible factor 1 (HIF-1), a pivotal transcription factor composed of HIF-1{alpha} and HIF-1ß subunits, plays a major role in tumor progression by activating a number of genes critically involved in adaptation to hypoxia. HIF-1 is also induced by several carcinogenic metals. Vanadate, an environmental toxic metal, is considered as a potent inducer of tumors in animals and is reported to activate HIF-1 activity. However, the involved mechanisms are poorly understood. In the present study, we have examined the biochemical mechanisms of the vanadate-induced HIF-1 activation in cancer cells by primarily focusing on the role of AMP-activated protein kinase (AMPK), which plays an essential role as an energy sensor under ATP-deprived conditions. We demonstrate that AMPK was rapidly activated in response to vanadate in DU145 human prostate carcinoma, and that its activation preceded HIF-1{alpha} expression. Under this condition, inhibition of AMPK by a pharmacological and molecular approach dramatically abolished the vanadate-induced HIF-1{alpha} expression as well as HIF-1-mediated physiological responses. Phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin signaling was also involved in vanadate-induced HIF-1{alpha} expression, but it was independent of AMPK signaling pathway. Moreover, we demonstrate a role of reactive oxygen species as an upstream signal for these two pathways. These results suggest that AMPK is a novel and critical component of HIF-1 regulation, further implying its involvement in vanadate-induced carcinogenesis.

Abbreviations: ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; AMPK, AMP-activated protein kinase; DCFH-DA, 2',7'-dichlorofluorescein-diacetate; DN, dominant negative; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Glut, glucose transporter; HIF-1, hypoxia-inducible factor 1; MAP, mitogen-activated protein; mTOR, mammalian target of rapamycin; PI, phosphatidylinositol; PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; ROS, reactive oxygen species; RT, reverse transcriptase; VEGF, vascular endothelial growth factor; WT, wild-type


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The reduced oxygen availability in aerobic organisms initiates a series of adaptive responses, and hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in these responses by transactivating a number of genes whose protein products function to enhance oxygen delivery and to facilitate anaerobic energy metabolism (1,2). The most prominent HIF-1 target genes are those encoding vascular endothelial growth factor (VEGF), erythropoietin, glucose transporters (Glut1 and 3) and glycolytic enzymes, which stimulates angiogenesis, erythropoiesis and anaerobic ATP production, respectively. HIF-1 is a heterodimer composed of HIF-1{alpha} subunit and HIF-1ß subunit proteins, which belongs to the basic helix–loop–helix PER-ARNT-SIM domain family of transcription factors. The functional activity of HIF-1 is primarily regulated by accumulation of the HIF-1{alpha} protein. Whereas the HIF-1ß subunit is constitutively expressed, the HIF-1{alpha} protein level is tightly controlled by the ambient oxygen tension. Under normoxic conditions, HIF-1{alpha} is continuously transcribed and translated, but its protein level is almost undetectable in most cells because of rapid degradation via the ubiquitin–proteasome system (1,2). A decline in oxygen tension blocks HIF-1{alpha} degradation and leads to its accumulation. In addition to protein stabilization, HIF-1 regulation includes post-translational modifications of HIF-1{alpha}, its nuclear translocation, and association with transcriptional cofactors.

Hypoxia is widespread in solid tumors, and tumor hypoxia is closely related with malignant progression, resistance to radiotherapy and chemotherapy, and increased metastasis (3). Accumulating data from cell culture, animal models and clinical studies indicate that many of these phenomena are associated with HIF-1 and its target gene products (4). Immunohistochemical studies indicate that HIF-1 is highly over-expressed in a variety of human cancers and that its level correlates with treatment failure and patient mortality (4). Furthermore, loss of HIF-1 directly resulted in a dramatic decrease in tumor growth, vascularization and energy metabolism in xenograft models (1,2). For these reasons, HIF-1 has been considered as a putative anticancer target. Thus, revealing mechanisms of the HIF-1 induction is clinically relevant and critical to understanding tumor pathogenesis. Although tissue hypoxia is a major inducer of HIF-1 activity, it is also activated by several carcinogenic metals such as nickel (5), chromium (6) and vanadate (7,8). Vanadium is a major trace metal in fossil fuels, and one of the main sources of the metal in human is through exposure to atmospheric vanadium, which is discharged by the combustion of the vanadium-bearing fuel oils (9). Vanadate or vanadate-containing compound shows potent carcinogenic effects on many biological systems; DNA damage, mutations, and DNA–protein crosslinks were induced by the metal in mammalian cells (10). The transforming activity of vanadate was also shown in mouse and hamster embryo cells (11,12). Furthermore, epidemiological evidence supports a correlation between chronic exposure to vanadate and increased risk of cancer (13). Nevertheless, the mechanism of the vanadate-induced HIF-1 activation is poorly understood. In the present study, we investigated the biochemical mechanisms of the vanadate-induced HIF-1 activation in human cancer cells by primarily focusing on the role of AMP-activated protein kinase (AMPK).

AMPK plays a major role in energy homeostasis by coordinating a number of adaptive responses under ATP-depleting metabolic stresses (14,15). AMPK, a heterotrimer consisting of a catalytic subunit ({alpha}) and two regulatory subunits (ß and {gamma}), is sensitively regulated by allosteric binding of AMP under pathological or physiological conditions causing ATP depletion such as ischemia/hypoxia, heat shock, oxidative stress and exercise. Upon activation, AMPK down-regulates several anabolic enzymes by phosphorylation and shuts down the ATP-consuming metabolic pathways, whereas its activation facilitates energy producing pathways (14,15). Previously, we demonstrated that AMPK activity is critical for HIF-1 transcriptional activity and its target gene expression under tumor hypoxia, suggesting that an energy-sensing signal is one of the essential components for the oxygen-regulated gene expression (16). Here, we investigated whether AMPK plays any role in vanadate-induced HIF-1 activation. In DU145 human prostate carcinomas, we examined the effect of vanadate on AMPK activity, a role of AMPK in HIF-1 induction, a cross-talk between AMPK and PI-3 kinase signal pathway, and a role of reactive oxygen species (ROS) as an upstream signal. Our results suggest that AMPK activity is critical for the vanadate-induced HIF-1 activation, further implying its novel role in metal-induced carcinogenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
RPMI medium 1640 and fetal bovine serum were purchased from Life Technologies. Sodium orthovanadate, dephostatin, LY294002, rapamycin and cycloheximide were obtained from Sigma. Compound C was a generous gift from Merck. 2-Deoxy-D-[3H]glucose (6.0 Ci/mmol) was purchased from Perkin-Elmer Life Sciences. The anti-phospho-specific antibodies that recognize phosphorylated ACC-Ser79, AMPK{alpha}-Thr172, mTOR (mammalian target of rapamycin)-Ser2448, Akt-Ser473 and p70S6K-Thr389 were from Cell Signaling Technology. Antibodies for HIF-1{alpha}, HIF-1ß and c-myc were purchased from Santa Cruz Biotechnology. AMPK{alpha} antibody was purchased from Upstate Biotechnology. Plasmid pEpoE-luc containing a HIF-1 binding site (5'-TACGTGCT-3') and pEpoEm-luc with a mutated site (5'-TAAAAGCT-3') were generously provided by Dr Franklin Bunn (Hematology-Oncology Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA).

Cell culture and protein extracts
DU145 (a human prostate carcinoma), HCT116 (a human colon carcinoma), AGS (a human gastric adenocarcinoma) and MCF-7 (a human breast carcinoma) were maintained in RPMI medium 1640 supplemented with 10% heat-inactivated fetal bovine serum and antibiotics at 37°C with 95% air and 5% CO2. Before exposure to vanadate, cells were incubated in serum-free media for 12 h, and then each reagent was added for the indicated time points. Total protein extracts were obtained using lysis buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin), and subjected to the western blot analysis.

Cycloheximide addition
The effect of the protein synthesis inhibitor cycloheximide on HIF-1{alpha} expression was examined as described previously (17). DU145 cells were exposed to vanadate (100 µM) or hypoxia for 12 and 4 h, respectively. Cycloheximide was then added to a final concentration of 100 µM and further incubated for 90 min in the presence of vanadate or under hypoxic condition. In case of hypoxia, cycloheximide addition was carried out inside a hypoxic chamber without disturbing oxygen tension. At different time points during cycloheximide addition, protein extracts were prepared and were subjected to immunoblot analysis.

Adenovirus-mediated gene transfer
AMPK wild-type {alpha}1 subunit (WT), a dominant negative form (DN) and a constitutively active form ({alpha}312) were generated by polymerase chain reaction as described previously (18). Recombinant adenovirus was prepared and purified as described previously (16). Infections with Ad-{alpha}1WT, Ad-{alpha}1DN or Ad-{alpha}312 were conducted at 100 plaque forming units (p.f.u.)/cell in phosphate-buffered saline for 30 min at 37°C, and then fresh serum-free medium was added for the indicated time periods.

RNA isolation and RT–PCR
Total RNA was extracted and cDNA was prepared as described previously (16). The cDNA fragment was amplified by PCR using the following specific primers: HIF-1{alpha}, sense 5'-CTTGCTCATCAGTTGCCACTT-3', anti-sense 5'GCCATTTCTGTGTGTAAGCAT-3'; VEGF, sense 5'-AGGAGGGCAGAATCATCACG-3', anti-sense 5'-CAAGGCCCACAGGGATTTTCT-3'; Glut-1, sense 5'-CGGGCCAAGAGTGTGAA-3', anti-sense 5'-TGACGATACCGGAGCCAATG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense 5'-TGCTGAGTATGTCGTGGAGTCTA-3', anti-sense 5'-AGTGGGAGTTGCTGTTGAAGTCG-3'; ß-actin, sense 5'-GTGGGGGCGCCCAGGCACCA-3', anti-sense 5'-CTCCTTAATGTCACGCACCATTTC-3'; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), sense 5'-ATTGGTCTGGCAACTGCAAA-3', anti-sense 5'-GGAGCCTCCTATGTGTGACT-3'. PCR was initiated in a thermal cycle programmed at 95°C for 5 min, 94°C for 1 min, 58°C for 1 min, 72°C for 1 min and amplified for 25 cycles. The amplified products were visualized on 1% agarose gels.

VEGF enzyme-linked immunosorbent assay (ELISA) assay
After exposure to vanadate for the indicated time period, the medium was removed and stored at –80°C until assayed. VEGF concentrations were determined using ELISA kit (R&D systems), following the manufacturer's instructions. Samples from three different experiments were analyzed in duplicate.

ROS measurement
Cells were incubated with 10 µM of 2',7'-dichlorofluorescein diacetate (Sigma) for 30 min, harvested by trypsinization, collected by centrifugation, washed with PBS and re-suspended in PBS containing 2 µg/ml propidium iodide. After sorting out the viable cells, fluorescence intensity was measured by flow cytometry (Becton-Dickinson) using excitation and emission wavelengths of 488 and 525 nm, respectively.

Reporter gene assays and glucose uptake
These assays were performed as described previously (16).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
AMPK is rapidly activated in response to vanadate in DU145 cells
We first examined the kinetics of AMPK activation and HIF-1 expression in DU145 human prostate carcinomas that were exposed to vanadate (100 µM) for the indicated time period (Figure 1A). The phosphorylation level of serine 79 of acetyl-CoA carboxylase (ACC), which is the best-characterized phosphorylation site by AMPK (14), was rapidly increased by vanadate treatment, reaching a peak within 1 h, as assessed by immunoblotting with an antibody specific for the phosphorylated ACC-Ser79 (Figure 1A). We have demonstrated previously the tight correlation between the phosphorylation level of ACC-Ser79 and endogenous AMPK activity (16). Furthermore, the phosphorylation level of Thr172 in the active site of AMPK{alpha} catalytic subunit, which is essential for the enzyme activity (19), was concomitantly increased. In the absence of any stimuli, the phosphorylation level of ACC-Ser79 and AMPKa-Thr172 remained basal at least for 12 h (Figure 7A). The total amount of ACC or AMPK{alpha} subunit was essentially the same. Therefore, these results indicate that AMPK was indeed activated by vanadate treatment. HIF-1{alpha} was detected at 8 h exposure to vanadate, whereas the expression level of HIF-1ß was not altered (Figure 1A). Vanadate also induced AMPK activation and HIF-1{alpha} expression in a dose-dependent manner, and maximum effect was observed at 100–200 µM (Figure 1B).



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Fig. 1. Effects of vanadate on AMPK activity and HIF-1 expression level in DU145 cells. DU145 cells were exposed to vanadate (100 µM) for the indicated time period (A) or exposed to different concentrations of vanadate for 12 h (B). Total cell extracts were prepared and subjected to western blot assay using anti-phosphospecific ACC-Ser79 (P-ACC), anti-ACC (ACC), anti-phosphospecific AMPK{alpha}-Thr172 (P-AMPK{alpha}), anti-AMPK{alpha} (AMPK{alpha}), anti-HIF-1{alpha} (HIF-1{alpha}) and anti-HIF-1ß (HIF-1ß) antibodies. (B) The phosphorylation level of ACC-Ser79 was examined after 1 h exposure to vanadate, whereas the protein level for HIF-1{alpha} and ß was determined after 12 h exposure, which respectively shows the maximum effect.

 


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Fig. 7. Two independent signal pathways, AMPK and PI-3 kinase/Akt/mTOR, are required for the vanadate-induced HIF-1{alpha} protein expression. (A) DU145 cells were incubated in the presence or absence of vanadate (100 µM) for 12 h. At the indicated time point, the phosphorylation level of ACC-Ser79 (P-ACC), AMPK{alpha}-Thr172 (P-AMPK{alpha}), Akt-Ser473 (P-Akt), mTOR-Ser2448 (P-mTOR) and p70S6K-Thr389 (P-p70S6K) as well as ACC protein level (ACC) were examined by immunoblot analysis using specific antibodies. (B) DU145 cells were pre-treated with compound C (20 µM), LY294002 (10 µM) or rapamycin (20 ng/ml) for 30 min, and then the effect of vanadate (100 µM) on the phosphorylation level of ACC-Ser79 and Akt-Ser473 as well as the protein level of HIF-1{alpha} (HIF-1{alpha}) and HIF-1ß (HIF-1ß) were examined by immunoblot analysis. (C) DU145 cells were infected with an increasing concentration of Ad-{alpha}1WT or Ad-{alpha}1DN (50, 100, 200 p.f.u.). Then, the effects of vanadate on the phosphorylation levels of ACC-Ser79, Akt-Ser473 as well as the protein level of HIF-1{alpha} were examined by immunoblot analysis. (B and C) The phosphorylation level of ACC-Ser79 and Akt-Ser473 was examined after 1- and 6-h exposure to vanadate, respectively, whereas the HIF-1{alpha} and ß levels were determined after 12-h exposure.

 
AMPK activity is required for vanadate-induced HIF-1 target gene expression and HIF-1 transcriptional activity
The AMPK activation apparently preceded HIF-1{alpha} induction in the vanadate-treated cells (Figure 1A), and such temporal profiles prompted us to examine whether AMPK activity is involved in vanadate-induced HIF-1 activity and its target gene expression (Figure 2). To this end, we first attempted to inhibit the AMPK activity either by a pharmacological approach or by a molecular approach and then examined the subsequent effect on the vanadate-induced HIF-1 target gene expression. Pre-treatment of DU145 cells with a specific AMPK inhibitor compound C (20), or adenovirus-mediated expression of c-myc-tagged AMPK DN form (Ad-{alpha}1DN) (18) almost completely prevented vanadate-induced AMPK activation in DU145 cells as indicated by the ACC-Ser79 phosphorylation level (Figure 2A). In a previous study, we indeed demonstrated that these two approaches effectively blunted the hypoxia-induced endogenous AMPK activity by performing the direct enzyme activity assay (16). Vanadate distinctively increased the mRNA level of HIF-1 target genes such as VEGF165, Glut1 and PFKFB3, as assessed by semi-quantitative RT–PCR, and such inductions were significantly abrogated by compound C or Ad-{alpha}1DN (Figure 2B). Under these conditions, the mRNA levels of GAPDH and ß-actin remained constant.



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Fig. 2. AMPK inhibition blocks the vanadate-induced VEGF165, Glut1 and PFKFB3 mRNA expression. (A) DU145 cells were either pre-treated for 30 min with compound C (20 µM), a pharmacological inhibitor of AMPK, or infected with Ad-{alpha}1WT or Ad-{alpha}1DN at 100 p.f.u./cell and incubated for an additional 24 h. Then, these cells were further exposed to vanadate (100 µM) for 1 h. Under these conditions, the phosphorylation level of ACC-Ser79 (P-ACC), total amount of ACC (ACC) and the expression level of the recombinant c-myc-tagged wild-type or dominant negative form of AMPK{alpha} (c-myc) were analyzed by western blot analysis using specific antibodies. (B) DU145 cells that were pre-treated with compound C or infected with Ad-{alpha}1WT or Ad-{alpha}1DN were exposed to vanadate (100 µM) for an additional 12 h. At each condition, the mRNA levels of VEGF165, Glut1, PFKFB3, GAPDH and ß-actin genes were examined by RT–PCR using specific primers. The amplified cDNA was analyzed on 1% agarose gel. Null, adenovirus with no exogenous gene; WT, Ad-{alpha}1WT; DN, Ad-{alpha}1DN.

 
In accordance with the transcript level (Figure 2B), the secreted VEGF protein amount, as measured by a commercial ELISA kit, increased ~4-fold in the culture media of DU145 cells that were exposed to vanadate for 24 h (Figure 3A). Under this condition, AMPK inhibition by compound C resulted in a significant abrogation of VEGF secretion. Likewise, vanadate-induced glucose uptake was also attenuated by compound C (Figure 3B). Consequently, these results (Figures 2 and 3) suggest that AMPK activity is required for vanadate-induced VEGF, Glut1 and PFKFB3 gene expression.



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Fig. 3. AMPK inhibitor prevents the vanadate-induced VEGF secretion and glucose uptake. (A) DU145 cells were pre-treated with compound C (20 µM) for 30 min, then exposed to vanadate (50 µM) for 24 h. Then, culture media were collected and the level of secreted VEGF was measured using a commercially available VEGF ELISA assay kit. (B) Under identical conditions, 2-deoxy-D-[3H]glucose was added to the culture media for 10 min at the end of the vanadate exposure period, and glucose uptake was measured. Results are the means ± SE of at least six determinations.

 
To determine whether AMPK modulates such gene expressions in HIF-1-dependent manner, we next investigated the effect of AMPK inhibition on HIF-1-dependent reporter gene expression (Figure 4). To this end, DU145 cells were co-transfected with a luciferase reporter pEpoE-luc containing a HIF-1-dependent hypoxia-responsive element (5'-TACGTGCT-3') and pcDNA3 expression vector encoding either AMPK wild-type {alpha}1 (AMPK-WT) or DN form {alpha}1 (AMPK-DN) (Figure 4A). As a negative control, pEpoEm-luc with a mutated site (5'-TAAAAGCT-3') was used. The transfected cells were exposed to vanadate for 12 h. Vanadate induced HIF-1-dependent luciferase activity ~7-fold, whereas the cells transfected with pEpoEm-luc showed no response (Figure 4A). Expression of AMPK DN form significantly diminished vanadate-induced HIF-1-dependent reporter gene expression (Figure 4A), and a more potent effect was observed when AMPK activation was inhibited by compound C (Figure 4B). Consequently, these results indicate clearly that AMPK activity is required for HIF-1 transcriptional activity and its target gene expression induced by vanadate.



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Fig. 4. Vanadate-induced HIF-1-dependent reporter gene expression requires AMPK activity. (A) DU145 cells were co-transfected with pEpoE-luc or pEpoEm-luc and pcDNA3 containing AMPK wild-type {alpha}1 subunit (AMPK-WT) or dominant negative {alpha}1 subunit (AMPK-DN) with 1:1 ratio. After 24 h post-transfection, cells were exposed to vanadate (100 µM) for an additional 12 h, and then luciferase activity was measured. (B) DU145 cells were transiently transfected with pEpoE-luc plasmid. After 24 h post-transfection, cells were exposed to vanadate (100 µM) for an additional 12 h in the presence or absence of compound C (20 µM). Then, cell lysates were subjected to the luciferase activity assay. The data represent means ± SE for six determinations.

 
AMPK activity is required for the vanadate-induced HIF-1{alpha} protein expression, but not for hypoxia-induced HIF-1{alpha} stabilization
To understand the underlying mechanisms of AMPK-mediated HIF-1 regulation, we next examined the effects of AMPK inhibition on HIF-1{alpha} protein level because the functional activity of HIF-1 is primarily regulated by accumulation of HIF-1{alpha} protein (1,2). In this experiment, we first compared the effect of AMPK inhibition on the vanadate- and hypoxia-induced HIF-1{alpha} expression. In DU145 cells, AMPK activation as well as HIF-1{alpha} expression is detected within 1 h under hypoxic condition (16). DU145 cells were pre-treated with increasing concentrations (20–40 µM) of compound C, and then exposed to vanadate or hypoxia (1% oxygen) for 12 and 4 h, respectively. At each condition, the expression levels of HIF-1{alpha} and HIF-1ß were examined by the western blot assay (Figure 5A). The results show that compound C inhibited the AMPK activity in a concentration-dependent manner, as indicated by the phosphorylation level of ACC-Ser79, leading to specific suppression of HIF-1{alpha} expression induced by vanadate, but not by hypoxia (Figure 5A, left panels). Essentially identical results were obtained when endogenous AMPK activity was abrogated by adenovirus-mediated expression of AMPK-DN (Figure 5A, right panels). The expression level of HIF-ß remained constant under all conditions. RT–PCR analysis shows that HIF-1{alpha} mRNA is expressed under normoxic conditions, which is consistent with the previous notion (1,2), and that its level was not altered by either vanadate or pre-treatment of compound C (Figure 5B), suggesting that vanadate may induce HIF-1{alpha} expression at a post-transcriptional level. These results further suggest that the mechanism for vanadate-induced HIF-1{alpha} expression should differ from a hypoxia-induced mechanism, and we reasoned that vanadate could stimulate protein synthesis of HIF-1{alpha}. To this end, the effect of cycloheximide, a protein synthesis inhibitor, on the kinetics of HIF-1{alpha} decay in DU145 cells exposed to vanadate or hypoxia was compared (Figure 5C). After exposure to vanadate (100 µM) or hypoxia for 12 and 4 h, respectively, DU145 cells were further incubated for 90 min in the presence of cycloheximide (100 µM). The protein level of HIF-1{alpha} in hypoxia-induced cells did not alter over 90 min in the presence of a protein synthesis inhibitor; supporting the previous notion that hypoxia stabilizes HIF-1{alpha} protein by blocking its degradation. In sharp contrast, HIF-1{alpha} in vanadate-treated cells rapidly degraded when protein synthesis was blocked by cycloheximide. The expression level of HIF-1ß remained constant under all conditions. Therefore, our results suggest collectively a possibility that vanadate induces HIF-1 activity via stimulating HIF-1{alpha} protein synthesis, which requires AMPK activity.



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Fig. 5. The differential effect of AMPK inhibition on HIF-1{alpha} induction in response to vanadate and hypoxia. (A) DU145 cells were pre-treated with an increasing concentration of compound C (20–40 µM) for 30 min (left panels), or infected with Ad-{alpha}1WT (WT) or Ad-{alpha}1DN (DN) (right panels). These cells were exposed to vanadate (100 µM) or hypoxia (1% oxygen) for 12 and 4 h, respectively, to determine the level of HIF-1{alpha} (HIF-1{alpha}) and HIF-1ß (HIF-1ß) by immunoblot analysis. The phosphorylation level of ACC-Ser79 (P-ACC) was determined in 1 h exposure to each stimulus. Exogenously introduced AMPK{alpha} was detected with c-myc antibody (c-myc). (B) DU145 cells were exposed to vanadate (100 µM) for 12 h in the presence or absence of compound C. At each condition, the mRNA levels of HIF-1{alpha}, GAPDH and ß-actin were examined by RT–PCR. (C) DU145 cells were exposed to vanadate (100 µM) or hypoxia for 12 and 4 h, respectively. Cycloheximide was added to a final concentration of 100 µM and further incubated for 90 min. Then, the protein level of HIF-1{alpha} and HIF-1ß was determined by immunoblot analysis.

 
We further examined the role of AMPK in several different human cancer cell lines including HCT116 colon carcinoma, AGS gastric adenocarcinoma and MCF-7 breast adenocarcinoma (Figure 6). Vanadate rapidly activated AMPK in these cell lines within 1 h, as indicated by the phosphorylation level of ACC-Ser79 and AMPK{alpha}-Thr172. HIF-1{alpha} expression was also detected in these cell lines in response to vanadate for 8 h, and AMPK inhibition by compound C distinctively abrogated HIF-1{alpha} expression, indicating that AMPK activity is likely to be necessary for the vanadate-induced HIF-1{alpha} expression in a broad range of cancer types.



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Fig. 6. The effect of AMPK inhibition on vanadate-induced HIF-1{alpha} protein expression in several cancer cell lines. HCT116, AGS and MCF-7 cells were treated with vanadate (100 µM) for 8 h in the absence or presence of compound C (20 µM). The phosphorylation levels of ACC-Ser79 (P-ACC) and AMPK{alpha}-Thr172 (P-AMPK{alpha}) were determined after 1 h exposure to vanadate, whereas the protein levels of HIF-1{alpha} (HIF-1{alpha}) and HIF-1ß (HIF-1ß) were examined after 8-h exposure to vanadate by western blot analysis.

 
PI-3 kinase/Akt/mTOR pathway is also required for the vanadate-induced HIF-1{alpha} protein expression, but it is independent of AMPK pathway
Many growth factors including insulin are known to stabilize HIF-1{alpha} under normoxic conditions, and recent reports demonstrated that activation of the PI-3 kinase/Akt/mTOR pathway by several growth factors could overcome the oxygen sensor-mediated HIF-1{alpha} degradation by stimulating HIF-1{alpha} protein translation rather than stability (17). Because vanadate shows various insulin-mimetic actions by activating PI-3 kinase pathway (21), we next attempted to elucidate the AMPK signaling pathway by checking a cross-talk with the PI-3 kinase/Akt/mTOR pathway (Figure 7). To this end, DU145 cells were incubated in the presence or absence of vanadate for the indicated time, and then we examined the kinetics of Akt, mTOR and p70S6K activation by determining the phosphorylation level of critical amino acid, which represents the activation of an upstream kinase (Figure 7A). While phosphorylation of ACC-Ser79 and AMPK{alpha}-Thr172 occurred relatively rapidly at 1 h exposure to vanadate, the phosphorylation level of Akt-Ser473, which indicates activation of upstream PI-3 kinase, was detected at 3 h, and maximum level was observed at 6 h exposure. Many growth factors such as insulin or insulin-like growth factor 1 stimulate mTOR kinase activity, which leads to several physiological events including activation of protein translation (22), and this effect is mediated in part by phosphorylation of mTOR-Ser2448 by Akt (23). The kinetic profile of phosphorylation of this residue upon exposure to vanadate showed a similar pattern as that of Akt-Ser473. One of the downstream targets of mTOR is p70S6K, and mTOR directly phosphorylates p70S6K on Thr389 (24). Vanadate also increased the phosphorylation level of p70S6K-Thr389 in a similar kinetic pattern as those of Akt-Ser473 and mTOR-Ser2448. Total amount of each tested kinase was not altered during the conditions (data not shown). Therefore, these results indicate that vanadate activated PI-3 kinase/Akt/mTOR signal pathway as well as AMPK signal pathway in our experimental condition.

To further evaluate the role of each signaling pathway on vanadate-induced HIF-1{alpha} expression, DU145 cells were pre-treated with pharmacological inhibitor for each kinase prior to exposure to vanadate, and then we examined the subsequent effects (Figure 7B). Compound C (AMPK inhibitor), LY294002 (PI-3 kinase inhibitor) or rapamycin (mTOR inhibitor) potently blunted the vanadate-induced HIF-1{alpha} expression, suggesting that at least two different signal pathways, AMPK and PI-3 kinase, are required for the process. Furthermore, AMPK inhibition by compound C has no effect on the vanadate-induced phosphorylation of Akt-Ser473. Conversely, LY294002 or rapamycin did not alter the vanadate-induced phosphorylation level of ACC-Ser79. Thus, both AMPK and PI-3 kinase/Akt/mTOR signal pathways are required for HIF-1{alpha} protein expression, but two pathways seem to transmit their signals in an independent manner. The role of AMPK in the vanadate-induced HIF-1{alpha} expression was re-examined by expressing the DN form of AMPK; the vanadate-induced HIF-1{alpha} expression was decreased as endogenous AMPK was inhibited by increasing the amount of AMPK-DN, but the phosphorylation level of Akt-Ser473 was not affected under the conditions (Figure 7C). These results indicate that vanadate activates at least two independent signal pathways, AMPK and PI-3 kinase/Atk/mTOR, which are required for HIF-1{alpha} expression.

Vanadate induces HIF-1{alpha} expression as well as activation of both AMPK and PI-3 kinase pathway via ROS generation
Based on the results of several previous reports, we reasoned that ROS could trigger upstream signals leading to both AMPK and PI-3 kinase activation in vanadate-induced HIF-1{alpha} expression. First, it is known that vanadate generates ROS, which plays a critical role in vanadate-induced carcinogenesis (25). Secondly, H2O2 activates PI-3 kinase pathways (26). Thirdly, we also demonstrated previously that AMPK is activated sensitively by H2O2 (27). For these reasons, the intracellular ROS level was examined in DU145 cells during exposure to vanadate, using 2',7'-dichlorofluorescein-diacetate (DCFH-DA) (Figure 8). DCFH-DA can be oxidized to DCF relatively specifically by hydrogen peroxide, resulting in increased fluorescence of the dye. DU145 cells were exposed to vanadate for 12 h. After additional incubation for 30 min in the presence of 10 µM DCFH-DA, we monitored changes in fluorescence intensity by fluorescence-activated cell scanning analysis. As shown in Figure 8A, vanadate markedly increased DCF fluorescence indicating ROS generation. Moreover, the fluorescence microscopic image of the cells under the identical conditions also indicated that vanadate dramatically generated ROS (Figure 8B). To demonstrate a direct role of ROS in the signaling pathway of the vanadate-induced HIF-1{alpha} expression, the effect of catalase, a specific H2O2 scavenger, was examined. Catalase treatment almost completely scavenged the vanadate-generated ROS (Figure 8A and B), resulting in an almost complete inhibition of the vanadate-induced HIF-1{alpha} expression, Akt activation and AMPK activation (Figure 8C). These results strongly suggest that ROS or H2O2 generated by vanadate induce HIF-1{alpha} expression by activating both the AMPK and PI-3 kinase/Akt pathway.



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Fig. 8. ROS generated by vanadate induces HIF-1{alpha} expression and activation of AMPK and Akt. DU145 cells were exposed to vanadate (100 µM) for 12 h in the presence or absence of catalase (5000 U/ml). (A) After an additional 30-min incubation in the presence of 10 µM DCFH-DA, changes in fluorescence intensity were measured by fluorescence-activated cell scanning analysis. (B) Under identical conditions, the confocal fluorescence microscopic images are presented. (C) After exposure to vanadate (100 µM) for 12 h in the presence or absence of catalase, the phosphorylation level of ACC-Ser79 (P-ACC), Akt-Ser473 (P-Akt) and protein levels of HIF-1{alpha} and HIF-1ß were examined.

 
Although our results support that vanadate acts as an oxidizing agent, we cannot completely rule out a possibility that the protein tyrosine phosphatase activity of vanadate is involved in AMPK and HIF-1 induction. To this end, we next compared the effects of another protein tyrosine phosphatase inhibitor dephostatin (28,29) and those of hydrogen peroxide on AMPK activity and HIF-1{alpha} expression (Figure 9). Dephostatin did not activate AMPK nor induce HIF-1{alpha} expression for 12 h (Figure 9A). Furthermore, intracellular production of ROS was not detected either under the condition (data not shown). In a sharp contrast, direct exposure of DU145 cells to hydrogen peroxide (300 µM) resulted in HIF-1{alpha} expression as well as rapid activation of AMPK (Figure 9A), and H2O2-induced HIF-1{alpha} expression was almost completely blocked by AMPK inhibitor (Figure 9B). Taken together, our results suggest that ROS generated by vanadate is a critical signal mediator for AMPK activation and subsequent induction of HIF-1{alpha} expression.



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Fig. 9. H2O2, but not dephostatin, induces HIF-1{alpha} expression via AMPK activation. (A) DU145 cells were treated with protein tyrosine phosphatase inhibitor dephostatin (100 µM) or exposed to H2O2 (300 µM) for 12 h. At the indicated time, protein extracts were prepared, and immunoblot analysis was performed using anti-phosphospecific ACC-{alpha} Ser79 (P-ACC), anti-phosphospecific AMPK-Thr172 (P-AMPK), anti-HIF-1{alpha} (HIF-1{alpha}) and anti-HIF-1ß (HIF-1ß) antibody. (B) DU145 cells were exposed to H2O2 (300 µM) for 4 h in the presence and absence of compound C (20 µM). Then, the phosphorylation level of ACC-Ser79 (P-ACC), and protein levels of HIF-1{alpha} and HIF-1ß were examined by immunoblot analysis.

 
AMPK activation alone is not sufficient to induce HIF-1{alpha} expression
Our results so far indicate that AMPK activity is necessary for HIF-1{alpha} protein expression in vanadate-treated cells. We next examined whether AMPK activation alone is sufficient to induce HIF-1{alpha} expression (Figure 10). For this reason, DU145 cells were treated with a cell permeable AMPK activator, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR). AICAR becomes a potent AMPK activator after its intracellular phosphorylation to AMP-mimetic AICA-ribotide (14,15). AMPK activation was sustained at least for 12 h by 1 mM AICAR, but HIF-1{alpha} was not detected during this period (Figure 10A). Furthermore, adenovirus-mediated expression of constitutive active form of AMPK (Ad-{alpha}312), which is a truncated N-terminal catalytic domain of the AMPK {alpha}1 subunit (18), did not induce HIF-1{alpha} expression either (Figure 10B), indicating that AMPK activation alone is not sufficient to induce HIF-1{alpha} expression.



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Fig. 10. The effect of a cell permeable AMPK activator AICAR and expression of constitutively active form of AMPK on HIF-1{alpha} expression. DU145 cells were either treated with AICAR (1 µM) for 12 h (A) or infected with adenovirus expressing the c-myc-tagged constitutively active form of AMPK (Ad-{alpha}312) (B). Immunoblot analysis was performed using anti-phosphospecific ACC-{alpha} Ser79 (P-ACC), anti-ACC (ACC), anti-HIF-1{alpha} and anti-c-myc antibody (c-myc).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several transition metals including vanadium can up-regulate HIF-1 and its target gene expression, but the molecular mechanism underlying the metal activation is almost unknown. An early report demonstrating that vanadate increased the basal level of HIF-1 proteins and HIF-1 activity has been considered as evidence implying the significance of phosphorylation for the regulation of HIF-1 activity because vanadate acts as a tyrosine phosphatase inhibitor (7). However, molecular mechanisms for this effect were not clearly delineated because vanadate also shows some other activities such as ROS production and various insulin-mimetic actions (21). In the present study, we have identified AMPK as a novel regulatory component for the vanadate-induced HIF-1 activation in cancer cells. Our data clearly show that blockade of AMPK activation by a pharmacological and molecular approach significantly abolished the vanadate-induced HIF-1 activation and the subsequent HIF-1-mediated responses. In a previous report, we also demonstrated that AMPK activity is essential for HIF-1 regulation under hypoxic conditions (16). Therefore, our results suggest that AMPK may play a critical role in HIF-1 regulation and its target gene expression under various conditions, further implying that AMPK may be also important to cancer development by regulating critical features of cancer cell adaptation such as angiogenesis and anaerobic glycolysis.

The protein levels and/or the transcriptional activity of HIF-1 can be induced in response to many growth factors and cytokines under normoxic conditions (1,2). In this induction, several receptor-mediated signal pathways were implicated, but the PI-3 kinase/Akt/mTOR pathway has received much attention lately as an alternative mechanism inducing HIF-1{alpha} expression under normoxic conditions (17); activation of this pathway by heregulin, insulin or IGF-1 increased the rate of HIF-1{alpha} protein synthesis rather than its stability, thereby overwhelming the oxygen-dependent degradation of HIF-1{alpha} (2). Our results suggest the mechanism for the vanadate-induced HIF-1{alpha} expression differs from the hypoxia-induced one because inhibition of protein synthesis showed dramatically different results between vanadate- and hypoxia-induced HIF-1{alpha} expression (Figure 5). It is well accepted that HIF-1{alpha} is stabilized under hypoxic conditions due to inhibition of the proteasome degradation pathway, and our results also support this notion because cycloheximide addition during hypoxia exerted no effect on the HIF-1{alpha} protein level (Figure 5C). In contrast, vanadate-induced HIF-1{alpha} expression seems to be dependent on protein synthesis because cycloheximide sensitively blocked HIF-1{alpha} expression in the presence of vanadate. Moreover, the involvement of the PI-3 kinase/Akt/mTOR pathway in the vanadate-induced HIF-1{alpha} expression (Figure 7) further supports this possibility, and this result is in good agreement with the previous report (8). Therefore, our data collectively suggest that AMPK positively regulate HIF-1{alpha} protein synthesis in response to vanadate, and its activity is likely to be required for the process in a variety of cancer cells (Figure 6). Moreover, the role of AMPK seems to be quite specific to HIF-1{alpha} expression because the protein levels of ACC, HIF-1ß (Figure 5A) and Akt (Figure 7) were not affected by AMPK inhibition. We have shown previously that AMPK regulate the HIF-1 transcriptional activity at the post-translational modification steps under hypoxic conditions (16). Therefore, depending on the environmental factors, AMPK may be differentially involved in multiple regulatory steps such as HIF-1{alpha} expression and its transcriptional activity, highlighting the significance of AMPK in HIF-1-mediated adaptive response. In fact, several signal molecules such as p44/42 MAP kinase, p38 MAP kinase or Rac1 can affect HIF-1{alpha} protein induction as well as HIF-1 transcriptional activity, depending on cell type and stimuli (3032).

Our results show that AMPK and PI-3 kinase/Akt/mTOR signal pathways play a similar role in the vanadate-induced HIF-1{alpha} expression. Thus, we further attempted to elucidate the underlying mechanisms by checking a cross-talk between two signaling pathways (Figure 7). AMPK is known as a master regulator of glucose and fatty acid metabolism in skeletal muscle, and it has been considered as a novel therapeutic target for the treatment of type 2 diabetes mellitus (15). Considering the well-known role of PI-3 kinase pathway in insulin action, there is indeed substantial interest in elucidating the cross-talk between these two signaling pathways. However, recent data on this issue are highly conflicting and controversial. Initial work on the role of AMPK in the control of glucose uptake suggested that AMPK activation promotes GLUT4 translocation to the plasma membrane via a PI-3 kinase-independent pathway (33). In contrast, several lines of evidence also suggest that AMPK functions upstream of Akt signaling (34), and another report shows that AMPK is activated in a PI-3 kinase-dependent manner when endothelial cells were exposed to oxidants such as ONOO (35). The opposite was found in a report demonstrating that Akt activation can lead to decreased AMPK activity in the heart (36). Furthermore, a recent report showing that AMPK activation by AICAR decreases Akt and mTOR activity (37) increases the complexity of understanding the cross-talk between these two signal pathways. Obviously, further work is required to elaborate the precise signaling mechanisms existing between these two pathways. However, our results presented in the current study support a possibility that these two signals are independent (Figure 7).

Vanadium can exist in oxidation states from –1 to +5, and ROS are generated during intracellular reduction of V(V) to V(IV) in the presence of NADPH and cellular enzymes such as NADPH oxidase (8,25). Our results show that vanadate distinctively generated ROS in DU145 cells and that catalase can completely abolish the vanadate-induced HIF-1{alpha} protein expression as well as activation of AMPK and PI-3 kinase pathway, implying the direct role of ROS in the process (Figure 8). In fact, ROS have been implicated in HIF-1 regulation (1,2). Considering that intracellular ROS can be generated by the numerous environmental factors and that AMPK is highly sensitive to oxidative stress (27), the role of AMPK may not be limited to vanadate-induced HIF-1{alpha} induction, but it may rather contribute to HIF-1 regulation under a variety of normoxic conditions accompanying ROS generation. For example, HIF-1 can be induced by various hormones or growth factors under normoxic conditions. Among these, HIF-1{alpha} induction by angiotensin II, thrombin and platelet-derived growth factors is known to be mediated through production of ROS in vascular smooth muscle cells (38). It was further suggested that these cells contain proteins that are similar to the components of NADPH oxidase, whose activation by these hormones leads to ROS production (38). In general, AMPK has been speculated to be stress-sensitive, but not hormone-sensitive. However, the possibility that AMPK is involved in HIF-1 regulation by hormones and growth factors mentioned above thus deserves further investigation, and such studies may broaden our understanding of a role of AMPK in a wide variety of cellular responses.


    Acknowledgments
 
This work was supported by Grant 02-PJ1-PG10-20904-0001 from the Ministry of Health and Welfare and Grant R13-2002-020-01004-0 from the Korea Science and Engineering Foundation through Medical Research Center for Bioreaction to Reactive Oxygen Species at Kyung Hee University.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 12, 2004; revised July 22, 2004; accepted July 25, 2004.





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