Nickel requires hypoxia-inducible factor-1alpha , not redox signaling, to induce plasminogen activator inhibitor-1

Angeline S. Andrew, Linda R. Klei, and Aaron Barchowsky

Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Human epidemiological and animal studies have associated inhalation of nickel dusts with an increased incidence of pulmonary fibrosis. At the cellular level, particulate nickel subsulfide inhibits fibrinolysis by transcriptionally inducing expression of plasminogen activator inhibitor (PAI)-1, an inhibitor of the urokinase-type plasminogen activator. Because nickel is known to mimic hypoxia, the present study examined whether nickel transcriptionally activates PAI-1 through the hypoxia-inducible factor (HIF)-1alpha signaling pathway. The involvement of the NADPH oxidase complex, reactive oxygen species, and kinases in mediating nickel-induced HIF-1alpha signaling was also investigated. Addition of nickel to BEAS-2B human airway epithelial cells increased HIF-1alpha protein levels and elevated PAI-1 mRNA levels. Pretreatment of cells with the extracellular signal-regulated kinase inhibitor U-0126 partially blocked HIF-1alpha protein and PAI-1 mRNA levels induced by nickel, whereas antioxidants and NADPH oxidase inhibitors had no effect. Pretreating cells with antisense, but not sense, oligonucleotides to HIF-1alpha mRNA abolished nickel-stimulated increases in PAI-1 mRNA. These data indicate that signaling through extracellular signal-regulated kinase and HIF-1alpha is required for nickel-induced transcriptional activation of PAI-1.

reduced nicotinamide adenine dinucleotide phosphate oxidase; nickel subsulfide; pulmonary fibrosis; mitogen-activated protein kinase; airway epithelium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INHALATION OF NICKEL DUST has been associated with an increased incidence of pulmonary fibrosis in humans and rodents (4, 9, 28, 35, 55). A possible mechanism for this effect of nickel was suggested to involve transcriptional induction of the plasminogen activator inhibitor (PAI)-1 gene in human airway cells (5). In these cells, nickel induced PAI-1 mRNA and protein levels, which led to chronic inhibition of fibrinolytic activity (5). The specific mechanisms driving the induction of PAI-1 by nickel are unknown but may involve the ability of nickel to mimic a response to hypoxia. Therefore, the present study investigated the hypothesis that nickel stabilizes the transcription factor hypoxia-inducible factor (HIF)-1alpha via the NADPH oxidase complex (purported to be an oxygen sensor), reactive oxygen species, or kinases, resulting in transcriptional activation of PAI-1.

HIF-1 is a heterodimeric basic helix-loop-helix-PAS transcription factor that consists of two subunits, HIF-1alpha and HIF-1beta (27). In normal physiology, low cellular oxygen concentrations increase HIF-1alpha protein levels and transcription of a number of genes. Hypoxia-inducible genes such as vascular endothelial growth factor (20), erythropoietin (34), glucose transporters, and glycolytic enzymes (52) are associated with increased oxygen delivery to tissues or metabolic adaptation to more anaerobic conditions (50). Hypoxia-induced increases in HIF-1alpha protein are thought to be due mainly to stabilization of the protein, which normally turns over with a 5-min half-life (30, 50).

Metals such as nickel, cobalt chloride, and mersalyl as well as the iron chelator deferoxamine also stabilize HIF-1alpha protein by unknown mechanisms. This stabilization leads to transcriptional activation of hypoxia-inducible genes (2, 11, 38, 44, 47, 49, 52). There are several theories to explain the ability of nickel to stabilize HIF-1alpha protein and to activate hypoxia-inducible gene expression. These models are based primarily on experiments done with other stimuli such as hypoxia, cobalt chloride, and deferoxamine. The major models involve the NADPH oxidase complex, a heme oxygen sensor, the mitochondrial electron transport chain, or kinase signaling. Reactive oxygen species are hypothesized to be central signaling molecules in several of the postulated mechanisms for the induction of HIF-1alpha by hypoxia and cobalt chloride (11, 27, 50, 51). Although several investigators (31, 32, 56) have noted increased production of reactive oxygen intermediates after nickel exposure, it is unclear whether this increase is involved in the stabilization of HIF-1alpha protein by nickel. Other studies indicate that kinases including diacylglycerol kinase (DAGK) (6), phosphatidylinositol 3-kinase (PI3K) (58), and extracellular signal-regulated kinase (ERK) (37, 45) are required for HIF-dependent transcription in response to hypoxia. The involvement of these kinases in the stabilization of HIF-1alpha and transcription of PAI-1 by nickel has not been previously investigated.

Nickel subsulfide is recognized as one of the more toxic forms of nickel because it is durable enough to be retained in the lung yet able to be dissolved inside lung cells (17, 18). Macrophages and epithelial cells ingest particles of nickel subsulfide into endocytic vesicles that fuse with acidic lysosomes. This acidification releases Ni2+, which reacts with cytoplasmic proteins (41) or heterochromatic regions of DNA (42, 53). The bound metal can then redox cycle to produce reactive oxygen species or affect protein conformations (36, 41). Therefore, nickel may activate cell signaling either through direct protein binding or through generation of reactive oxygen species.

In rat hepatocytes, hypoxia-induced PAI-1 expression is mediated by hypoxia-responsive elements in the promoter (43). This study identified both a strong and a weak hypoxia response element (HRE) that binds HIF-1alpha under conditions of hypoxia (43). Demonstration of active HREs in the promoter of the human gene has not been reported, and the human sequence shows homology only with the weak element in the rat promoter. Thus, although these data are supportive, the role of HIF-1alpha in nickel induction of the human PAI-1 gene is unresolved. This present study was designed to investigate the potential mechanisms for nickel-induced stabilization of HIF-1alpha and to determine the involvement of this transcription factor in the transcriptional activation of PAI-1 by nickel. Results of this investigation indicate that increases in HIF-1alpha protein levels are critical to the ability of nickel to induce PAI-1. Furthermore, the subcellular mechanism driving stimulation of the hypoxia-responsive pathway leading to PAI-1 activation by nickel may involve a kinase such as ERK but does not seem to be affected by changing the redox status of the cell or inhibiting the NADPH oxidase complex or the mitochondrial electron transport chain. These data increase the understanding of mechanisms for nickel-induced PAI-1 transcription, which may be critical for understanding the pathology of pulmonary fibrosis, cancer, and other serious diseases associated with nickel exposure.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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Cells. Human bronchial epithelial cells (BEAS-2B; American Type Culture Collection, Manassas, VA) were grown to postconfluence in 6- or 12-well plates (Corning Costar, Corning, NY) on a matrix of 0.01 mg/ml of human fibronectin (Collaborative Biomedical Products, Bedford, MA), 0.03 mg/ml of Vitrogen 100 (Collagen Biomaterials, Palo Alto, CA), and 0.01 mg/ml of bovine serum albumin (Sigma, St. Louis, MO). The cultures were maintained in LHC-9 medium (Biofluids, Rockville, MD) at 37°C under an atmosphere of 5% CO2-95% air. The cells were subcultured with 0.1% trypsin-EDTA and plated in tissue culture plates.

Nickel. The respirable-size fraction of nickel used in these experiments was prepared by applying nickel subsulfide (Ni3S2) particles (Aldrich, Milwaukee, WI) to a water column and allowing the larger particles to settle out. Particle size was measured during settling with a particle counter (Coulter, Miami, FL). Nickel subsulfide particles <2.5 µm in diameter were decanted, concentrated by centrifugation, and sterilized by baking at 200°C for 18 h. This preparation gives the same quantitative and qualitative responses as a standard preparation of nickel subsulfide obtained from the Nickel Producers Environmental Research Association (Durham, NC; a kind gift from Dr. Andrea Oller).

Treatments. As shown by a clonogenic survival assay, the addition of 2.34 µg Ni/cm2 of nickel subsulfide is not toxic to this cell model (5). In the present study, the cells were treated for up to 24 h with 0.58-2.34 µg Ni/cm2 (5). Deferoxamine mesylate (260 µM) was applied as a positive control to induce HIF-1alpha protein levels. Phorbol 12-myristate 13-acetate (PMA; 2-100 nM) was used as a positive control to induce PAI-1 expression. Cells were pretreated with 2-10 mM N-acetyl-L-cysteine (NAC), 2 mM ascorbic acid, 2 mM superoxide dismutase (SOD), 5 mM apocynin (Aldrich), 5 mM rotenone, 10 µM diphenyleneiodonium chloride (DPI), 20 µM SB-203580 (Calbiochem, La Jolla, CA), 10 µM U-0126 (Calbiochem), 1 µM wortmannin (Calbiochem), or 10 µM DAGK inhibitor (DAGKI) R-59949 (Calbiochem). Dimethyl sulfoxide (DMSO) was added as a vehicle control for treatment with DPI (0.01 µl/ml) or R-59949 (0.1% vol/vol). All reagents mentioned above were purchased from Sigma unless otherwise noted.

mRNA levels. Total cellular RNA was harvested with TRIzol reagent (GIBCO BRL, Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions and quantitated by spectrophotometric absorbance at 260 nm. Reverse transcription-polymerase chain reaction (RT-PCR) was performed as previously described by Barchowsky et al. (8) with PAI-1 (forward, 5'-cgcctcttccacaaatcag-3' and reverse, 5'-atgcgggctgagactatga-3'), beta -actin, or HIF-1alpha (forward, 5'-tcaccacaggacagtacaggatgc-3'and reverse, 5'-ccagcaaagttaaagcatcaggttcc-3') specific primers that were synthesized in the Molecular Biology Core at Dartmouth University (Hanover, NH) and reagents from Ambion (Austin, TX), Promega (Madison, WI), and Amersham Pharmacia Biotech (Piscataway, NJ). PCR products were either run on agarose gels stained with ethidium bromide or quantified with the double-strand DNA fluorescent dye PicoGreen (Molecular Probes Eugene, OR) at 430-nm emission and 525-nm absorption. Densitometry was performed on ethidium bromide stained gels with National Institutes of Health Image software. PAI-1 mRNA expression was normalized to the housekeeping gene beta -actin by taking the ratio of the PAI-1 to beta -actin band density or PicoGreen fluorescence.

2',7'-Dichlorofluoroscein fluorescence. 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluoroscein diacetate (CM-DCFH-DA) was purchased from Molecular Probes. Ninety-six-well plates of cells were incubated for 30 min to 24 h with antioxidants in 100 µl of LHC-9 medium. CM-DCFH-DA was prepared in LHC-9 medium and added to the cells at a final concentration of 20 µM in 200 µl/well. The cells were incubated with 2.34 µg Ni/cm2 for 10 min at 37°C, and the plates were read on a microplate fluorescence reader with excitation at 485 nm and emission at 530 nm. Similar results were obtained when the cells were treated with nickel for 24 h before the addition of CM-DCFH-DA.

Protein levels. The effects of nickel on HIF-1alpha or beta -actin protein levels were determined by Western blotting with a polyclonal antibody to HIF-1alpha (Transduction Laboratories, Lexington, KY) or a monoclonal antibody to beta -actin (Sigma). Immunoblotting was essentially as described by Barchowsky et al. (7). Briefly, at the end of exposure periods, all cells were placed on ice and rinsed with ice-cold stop buffer (10 mmol/l of Tris · HCl, pH 7.4, 10 mmol/l of EDTA, 5 mmol/l of EGTA, 100 mmol/l of NaF, 200 mmol/l of sucrose, 100 µmol/l of sodium orthovanadate, 5 mM sodium pyrophosphate, 4 µg/ml of leupeptin, 4 µg/ml of soybean trypsin inhibitor, 1 mmol/l of benzamidine, 20 µmol/l of calpain inhibitor-1, 100 mU/ml of aprotinin, and 100 µmol/l of phenylmethylsulfonyl fluoride). The stop buffer was replaced with a minimal volume of 2× SDS-PAGE buffer. The proteins were separated by PAGE and transferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA), and the membranes were blocked with 5.0% milk in 0.01 mol/l of Tris, pH 8.0, 0.15 mol/l of NaCl, and 0.05% Tween 20. Primary antibodies were added for either 1 h at room temperature or 24 h at 4°C. Secondary sheep anti-mouse IgG conjugated to horseradish peroxidase (Amersham, Arlington Heights, IL) was added for 30 min, and antibody complexes were detected by enhanced chemiluminescence (Renaissance, NEN Life Science, Boston, MA).

Antisense oligonucleotides. HIF-1alpha phosphorothioate antisense and sense oligonucleotides were synthesized in the Molecular Biology Core at Dartmouth University according to sequences published by Caniggia et al. (12). The cells were incubated for 24 h with 10 µM sense or antisense oligonucleotide before nickel treatment.

Statistics. Statistical analysis was performed on data pooled from duplicate or triplicate determinations in two to three separate experiments to yield a total of 6 experiments. Significant differences between treatment and control groups were determined with one-way analysis of variance. The means of groups were compared with Newman-Keuls post hoc test. All statistics were performed with GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA). Data are presented as means ± SD or as percentages of control values.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Time course and dose response of nickel subsulfide-induced HIF-1alpha in BEAS-2B cells. Nickel disrupts the fibrinolytic cascade by inducing transcription of PAI-1 (5). To investigate the hypothesis that nickel increases the levels of HIF-1alpha to induce transcription of the PAI-1 gene, we first examined the time course for nickel induction of HIF-1alpha protein levels in our cell model. Exposure of postconfluent BEAS-2B cells to 2.34 µg Ni/cm2 for up to 24 h increased HIF-1alpha protein levels beginning at 4 h, with maximal increases at 24 h. The Western analysis shown in Fig. 1A demonstrated that doses as low as 0.058 µg Ni/cm2 could increase HIF-1alpha protein at the 8-h time point. In contrast, HIF-1alpha mRNA levels were not induced and, in fact, were significantly decreased after 15 h of nickel treatment (Fig. 1, B and C). Thus increased protein stabilization appears to be the primary explanation for the dose- and time-dependent induction of HIF-1alpha protein levels induced by nickel.


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Fig. 1.   Nickel induces hypoxia-inducible factor (HIF)-1alpha protein. A: confluent cells exposed to 2.34 µg Ni/cm2 of nickel subsulfide (Ni3S2) for 2, 4, 8, or 24 h or 260 µM deferoxamine (DFO) for 8 h. The dose-response relationship was tested for 8 h by exposing cells to concentrations of nickel subsulfide ranging from 0.058 to 2.34 µg Ni/cm2. Total protein was harvested in 2× SDS buffer, and Western blots were performed as described in METHODS with an antibody to HIF-1alpha . ctrl, Control. Data represent protein collected from separate cultures and are representative of at least 5 replicates. B: total RNA was isolated from cells exposed to 2.34 µg Ni/cm2 for indicated times. HIF-1alpha and beta -actin mRNA levels were measured by RT-PCR and ethidium bromide staining. C: ratio of HIF-1alpha to beta -actin density of ethidium bromide-stained bands of PCR products in ultraviolet-transluminated 2% agarose gels. PMA, phorbol 12-myristate 13-acetate. Values are means ± SD; n = 3 experiments. *** P < 0.001 vs. respective control.

Antioxidants inhibit increases in intracellular oxidations induced by nickel. CM-DCFH-DA fluorescence was used to confirm that nickel subsulfide induced intracellular reactive oxygen species (31, 32, 56) in the BEAS-2B cells. The cells were left untreated or were pretreated with 10 mM NAC or 2 mM ascorbic acid before the addition of 20 µM CM-DCFH-DA and nickel subsulfide. The results in Fig. 2 support previous reports from other cell types (31, 48) that nickel increases CM-DCFH-DA fluorescence and that these increases can be blocked by pretreatment with antioxidants such as NAC and ascorbic acid.


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Fig. 2.   Nickel-induced 2',7'-dichlorofluoroscein (DCF) fluorescence is blocked by antioxidants. Confluent cells were pretreated with 10 mM N-acetyl-L-cysteine (NAC) for 18 h or 2 mM ascorbic acid for 15 min. Cells were loaded with 20 µM DCF for 10 min before 2.34 µg Ni/cm2 exposure. Fluorescence was measured with a fluorescence plate reader as described in METHODS. +, Presence; -, absence. Data were collected from separate cultures and are representative of at least 10 replicates; n = 3 experiments. *** P < 0.01.

Antioxidants, NADPH oxidase, and mitochondrial inhibitors do not block nickel-induced HIF-1alpha protein. Reactive oxygen species, the mitochondrial electron transport chain, and the NADPH oxidase complex are potentially involved in mechanisms that stabilize HIF-1alpha protein in response to nickel, cobalt chloride, and hypoxia (11, 50, 51). Therefore, the ability of NADPH oxidase inhibitors, mitochondrial electron transport chain inhibitors, or antioxidants to inhibit nickel-induced HIF-1alpha protein levels was determined. In Fig. 3A, confluent cells left untreated or pretreated with NAC, apocynin, or rotenone were exposed to nickel subsulfide for 24 h. The HIF-1alpha immunoblot in Fig. 3A shows that neither the antioxidant NAC nor the mitochondrial inhibitor rotenone blocked nickel-induced HIF-1alpha protein. Although the apocynin treatment decreased the intensity of the nickel-induced HIF-1alpha band, the concomitant decrease in basal HIF-1alpha levels suggested that this may not be a specific block. Figure 3B shows that increasing the dose of the antioxidant NAC to 10 mM still did not inhibit the induction of HIF-1alpha protein by nickel. In fact, NAC seemed to have a stimulatory effect on HIF-1alpha expression. The HIF-1alpha immunoblot in Fig. 3C confirms that pretreatment with the NADPH oxidase inhibitors apocynin and DPI did not block HIF-1alpha responsiveness to nickel subsulfide. There was no effect of adding 0.01 µl/ml of DMSO, the vehicle for DPI treatment.


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Fig. 3.   Antioxidants and NADPH oxidase inhibitors do not block nickel-induced HIF-1alpha protein. A: confluent cells left untreated or pretreated with 2 mM NAC for 18 h, 5 mM apocynin (Apo), or 5 mM rotenone (Roten) for 30 min were exposed to 2.34 µg Ni/cm2 for 24 h. B: confluent cells left untreated or pretreated with NAC for 18 h were exposed to 2.34 µg Ni/cm2 or 2 nM PMA for 24 h. C: confluent cells left untreated or pretreated with 5 mM apocynin or 10 µM diphenyleneiodonium (DPI) for 30 min were exposed to 2.34 µg Ni/cm2, 260 µM deferoxamine, 100 nM PMA, or 0.01 µl/ml of DMSO. Untreated cells of the same passage number were used as controls. Cells were harvested in 2× SDS buffer, and Western blots were performed with an antibody to HIF-1alpha followed by beta -actin as a loading control. Data represent protein collected from separate cultures and are representative of at least 6 replicates.

Antioxidants and NADPH oxidase inhibitors do not block induction of PAI-1 mRNA by nickel. To investigate whether reactive oxygen species and the NADPH oxidase complex mediate nickel induction of PAI-1 mRNA, cells pretreated with NAC, ascorbic acid, SOD, or apocynin were exposed to nickel subsulfide or deferoxamine (a positive control for activating HIF-1alpha ) for 24 h. As seen in Fig. 4A, antioxidants do not block nickel-induced PAI-1 mRNA levels. The NADPH oxidase inhibitor apocynin alone seemed to stimulate PAI-1 mRNA levels; however, apocynin pretreatment did not block nickel-induced PAI-1 (Fig. 4B). Deferoxamine, which is known to promote HIF-1alpha responses, induced a significant increase in PAI-1 mRNA levels.


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Fig. 4.   Antioxidants and NADPH oxidase inhibitors do not block induction of plasminogen activator inhibitor (PAI)-1 mRNA by nickel. A: pretreatment of cells with NAC for 18 h, 300 U/ml of superoxide dismutase (SOD), or 2 mM ascorbic acid for 30 min was followed by 24 h of exposure to 2.34 µg Ni/cm2 or no treatment. Total RNA was collected, and PAI-1 and beta -actin mRNA levels were determined by RT-PCR followed by staining with ethidium bromide. B: untreated cells or cells pretreated with 5 mM apocynin for 30 min were exposed to 2.34 µg Ni/cm2, 0.01 µl/ml of DMSO (crosshatched bars), or 260 µM deferoxamine for 24 h. Untreated cells of the same passage number were used as controls. Total RNA was collected, and PAI-1 and beta -actin mRNA levels were determined by RT-PCR followed by staining with PicoGreen double-strand DNA dye. Data represent RNA collected from separate cultures and are representative of at least 5 replicates. *** P < 0.001.

HIF-1alpha antisense oligonucleotide blocks nickel-induced HIF-1alpha protein. HIF-1alpha antisense oligonucleotide was used to investigate the hypothesis that HIF-1alpha is necessary for PAI-1 transcriptional activation by nickel. To verify the effectiveness of the HIF-1alpha antisense oligonucleotide, it was added for up to 56 h followed by a 6-h treatment with nickel subsulfide. HIF-1alpha sense oligonucleotide and PMA were added for 24 h as a control. The immunoblot in Fig. 5 shows that the HIF-1alpha antisense oligonucleotide effectively inhibited nickel-induced HIF-1alpha protein levels at all time points tested. The HIF-1alpha sense oligonucleotide slightly elevated HIF-1alpha protein levels in these cells.


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Fig. 5.   HIF-1alpha antisense oligonucleotide blocks nickel-induced HIF-1alpha protein. A: cells were pretreated with 10 µM HIF-1alpha antisense oligonucleotide for 3-56 h before 6 h of exposure to 2.34 µg Ni/cm2. Untreated cells or cells exposed to 10 µM HIF-1alpha sense oligonucleotide for 24 h were used as controls. Cells were harvested in 2× SDS buffer, and Western blots were performed with an antibody to HIF-1alpha . Data are representative of at least 6 replicates from separate cultures.

HIF-1alpha antisense but not sense oligonucleotide blocks nickel-induced PAI-1 mRNA. To determine whether inhibiting HIF-1alpha nickel responsiveness with an antisense oligonucleotide would inhibit induction of the endogenous PAI-1 gene, cells were pretreated with HIF-1alpha antisense or sense oligonucleotide followed by 6 h with nickel subsulfide. Only the antisense oligonucleotide blocked the ability of nickel but not of PMA to stimulate PAI-1 mRNA levels (Fig. 6, A and B). In contrast, treatment with the HIF-1alpha sense oligonucleotide had no effect on the induction of PAI-1 by nickel (Fig. 6, C and D).


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Fig. 6.   HIF-1alpha antisense but not sense oligonucleotide blocks nickel-induced PAI-1 mRNA. A: cells were pretreated with 10 µM HIF-1alpha antisense oligonucleotide for 24 h before 24 h of exposure to 2.34 µg Ni/cm2 or 100 nM PMA. Untreated cells or cells exposed to 10 µM HIF-1alpha sense oligonucleotide for 24 h were used as controls. Total RNA was collected, and PAI-1 and beta -actin mRNA levels were determined by RT-PCR followed by ethidium bromide staining. B: ratio of PAI-1 to beta -actin density of ethidium bromide-stained bands of PCR product in ultraviolet-transluminated 2% agarose gels of treated cells. Values are means ± SD of densitometric analysis of results in A from 2 pooled experiments, each performed in triplicate, for a total of 6 experiments. Significantly different from control value: *** P < 0.001; ** P < 0.01. C: cells were pretreated with 10 µM HIF-1alpha sense oligonucleotide for 24 h before 24 h of exposure to 2.34 µg Ni/cm2. Total RNA was harvested, and RT-PCR was performed with primers to PAI-1 or beta -actin mRNA. Bands of PCR product were ethidium bromide-stained in ultraviolet-transluminated 2% agarose gels. D: PCR products, including those in A, were stained with PicoGreen double-strand DNA stain. Data are means ± SD of ratio of PAI-1 to beta -actin fluorescence after PicoGreen staining and are representative of at least 6 replicates from separate cultures. *** P < 0.001 vs. respective control value.

Upstream kinases may be involved in the stabilization of HIF-1alpha by nickel. Reports in the literature (6, 14, 58) suggest that phosphorylation of HIF-1alpha by several upstream kinases may be involved in the transactivation of HIF-driven genes. Cells were pretreated with kinase inhibitors followed by 24 h of treatment with nickel subsulfide to determine whether these kinases were involved in the stabilization of HIF-1alpha by nickel. The ERK inhibitor U-0126 partially inhibited nickel-stimulated HIF-1alpha protein levels, whereas the DAGKI had a less dramatic inhibitory effect (Fig. 7). In contrast, the p38 inhibitor SB-20358 and the PI3K inhibitor wortmannin did not decrease nickel-induced HIF-1alpha protein levels.


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Fig. 7.   Kinases may be involved in the induction of PAI-1 by nickel. Cells were pretreated for 90 min with 1 µM wortmannin (Wort) or for 30 min with 20 µM SB-203580 (SB), 10 µM U-0126, or 10 µM diacylglycerol kinase inhibitor R-59949 (DAGKI) followed by 24 h of exposure to 2.34 µg Ni/cm2. DMSO (0.1% vol/vol) was added as the vehicle control, and 260 µM DFO was added as a positive control for HIF-1alpha induction. Cells were harvested in 2× SDS buffer, and Western blots were performed with an antibody to HIF-1alpha . Data are representative of at least 3 replicates from separate cultures.

Kinases may be involved in the induction of PAI-1 by nickel. PAI-1 mRNA levels were compared in cells that were pretreated with and without kinase inhibitors to determine whether kinases were involved in the transcriptional activation of PAI-1 by nickel. U-0126 caused the most significant inhibition of nickel-induced PAI-1 mRNA. SB-20358, wortmannin, and the DAGKI all had modest effects on nickel-stimulated PAI-1 mRNA levels.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to test the hypothesis that the induction of PAI-1 mRNA by nickel was mediated by the transcription factor HIF-1alpha . The mechanisms for stimulating increases in HIF-1alpha protein levels have been controversial and may be stimulus specific. The actions of nickel are further complicated because it has the paradoxical actions of promoting increased cellular redox cycling and mimicking hypoxia. The data presented in Fig. 2 confirm that nickel increases intracellular oxidation, but neither these oxidants, the mitochondrial electron transport chain, nor the NADPH oxidase complex mediate nickel-induced HIF-1alpha protein levels or expression of PAI-1. Eliminating HIF-1alpha from the cells, however, prevents the nickel-induced transcription of PAI-1 mRNA (Fig. 6). Pretreatment with kinase inhibitors, particularly U-0126, inhibits both the stabilization of HIF-1alpha by nickel as well as the ability of nickel to induce PAI-1 mRNA.

Reactive oxygen species are hypothesized to be central signaling molecules in several different postulated mechanisms for the induction of HIF-1alpha by hypoxia and cobalt chloride (11, 27, 50, 51). The generation of free radicals by nickel (31, 32, 56) has been cited as a mechanism for the stimulation of other transcription factors such as nuclear factor-kappa B and genes including intercellular adhesion molecule-1 (24) and interleukin-1 (15). If changes in reactive oxygen levels were critical for nickel-induced stabilization of HIF-1alpha protein, one would expect that the addition of antioxidants would prevent nickel from increasing HIF-1alpha protein levels. In contrast, the data in Fig. 3 indicate that pretreatment with a variety of antioxidants including NAC, which increases intracellular glutathione levels (19), or ascorbic acid, which scavenges reactive oxygen species (46), were completely ineffective in inhibiting nickel-induced HIF-1alpha protein levels. As shown in Fig. 2, these concentrations of antioxidants blocked the increases in intracellular oxidants by nickel. These results indicate that changes in reactive oxygen species levels are not necessary for nickel signaling in the HIF-1alpha -PAI-1 pathway. This finding supports a study by Salnikow et al. (49) showing that induction of another hypoxia-responsive gene, Cap43, by nickel was also not mediated by reactive oxygen species. Furthermore, the fact that HIF-1alpha is stabilized in the presence of the enhanced oxidative state after nickel exposure argues against the hypothesis that a decrease in oxidants is required to activate HIF-1alpha (Fig. 1).

The mitochondrial electron transport chain is also postulated to be involved in the hypoxic signaling cascade. Even though the mitochondrial electron transport chain complex I inhibitor rotenone does block the induction of HIF-1alpha protein in response to hypoxia, it does not affect HIF-1alpha expression induced by cobalt chloride or deferoxamine (3, 13). In the present study, pretreatment with rotenone failed to inhibit the HIF-1alpha response to nickel (Fig. 3A), indicating that mitochondrial electron transport is probably not involved in the induction of HIF-1alpha by this metal.

Other models of hypoxia-stimulated signal transduction indicate the involvement of a NADPH oxidase complex. The oxidants produced by this complex act as chemical messengers, mediating increases in HIF-1alpha protein stabilization (1, 23). If the NADPH oxidase complex were critical to the induction of HIF-1alpha by nickel, one would expect that NADPH oxidase inhibitors would block this induction. The data in Fig. 3C do not support this hypothesis. Treatment with apocynin, which inhibits NADPH oxidase by blocking the SH3 domain on the p47phox subunit, preventing it from interacting with the complex (33, 54, 57), failed to block nickel-induced HIF-1alpha protein. Likewise, treatment with DPI, an inhibitor of electron transfer through the gp91 subunit of the NADPH oxidase complex (40), also did not inhibit the induction of HIF-1alpha by nickel. Thus it is unlikely that this complex or a change in the oxidants generated by the complex is necessary for the nickel induction of HIF-1alpha (Fig. 3C).

Another possible mechanism for the action of nickel and the inability of NAC to protect against increases in HIF-1alpha protein levels might involve a heme oxygen sensor (11). According to this model, a lack of oxygen bound to the heme center stimulates a signaling cascade, which ultimately stabilizes the HIF-1alpha protein (22, 25, 26, 29). Nickel is unique among metals in its ability to interact with iron-sulfur centers. Metals like cobalt and nickel or iron chelators like deferoxamine might substitute for the iron in the center of the heme or prevent oxygen binding, triggering the hypoxia response pathway (11, 44, 50). It is also possible that nickel reacts directly with oxidized sulfur in the center to cause activation. NAC would not be expected to protect against this type of reaction.

Nickel may stimulate proximal events in signaling cascades, such as the insulin-like growth factor receptor, following a pathway similar to that activated by mersalyl, an organic mercury compound (50). Reports in the literature (10, 37, 45) indicate that the transactivation of HIF by hypoxia may require phosphorylation by ERK. Involvement of ERK in nickel signaling was supported by the data in Figs. 7 and 8 showing that treatment with U-0126 partially blocked the stabilization of HIF-1alpha and the induction of PAI-1 mRNA by nickel. ERK also phosphorylates other transcription factor binding proteins including c-Jun, which binds to activator protein-1 sites (16). Cooperativity between HIF-1alpha and activator protein-1 is required for hypoxia to induce several genes, including lactate dehydrogenase, erythropoietin, and tyrosine hydroxylase (21, 39), and could potentially be involved in the PAI-1 promoter.


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Fig. 8.   Kinases may be involved in the induction of PAI-1 by nickel. A: cells were pretreated for 90 min with 1 µM wortmannin or for 30 min with 20 µM SB-203580 or 10 µM U-0126 followed by 24 h of exposure to 2.34 µg Ni/cm2. Total RNA was collected, and PAI-1 and beta -actin mRNA levels were determined by RT-PCR followed by ethidium bromide staining. C: cells were pretreated with 10 µM DAGKI followed by 24 h of exposure to 2.34 µg Ni/cm2. DMSO (0.1% vol/vol) was added as the vehicle control, and 10 nM PMA was added as a positive control for PAI-1 mRNA induction. B and D: ratios of PAI-1 to beta -actin density of ethidium bromide-stained bands of PCR product in ultraviolet-transluminated 2% agarose gels of treated cells compared with control values. Data are means ± SD of densitometric analysis of results in A and C, respectively, from 2 pooled experiments, each performed in triplicate, for a total of 6 experiments. *** P < 0.001 vs. respective control.

The involvement of PI3K and signaling through DAGK to increase phosphatidic acid (6) have also been reported to mediate stabilization of HIF-1alpha in response to hypoxia. As shown in Fig. 7, treatment with the DAGKI R-59949 partially inhibited stabilization of HIF-1alpha by nickel but caused a modest decrease in nickel-stimulated PAI-1 mRNA (Fig. 8, C and D). These data indicate that ERK and possibly DAGK are involved in the upstream signaling pathways leading to the stabilization of HIF-1alpha and the induction of PAI-1 mRNA by nickel.

The hypothesis that increased levels of the transcription factor HIF-1alpha could be mediating nickel-induced transcription of the PAI-1 gene was supported by the data in Fig. 1 indicating that HIF-1alpha protein levels and PAI-1 mRNA levels are increased after a similar time course of nickel exposure. The increases start at 4 h, with the highest HIF-1alpha and PAI levels detectable at 24 h (5). HIF-1alpha antisense oligonucleotide dramatically inhibited the induction of PAI-1 mRNA levels by nickel (Fig. 6). These data implicate HIF-1alpha as a critical transcription factor involved in the nickel induction of PAI-1 gene transcription. Data in Fig. 4B further support the importance of HIF-1 to activation of the PAI-1 promoter because treatment with another agent that increases HIF-1alpha levels, deferoxamine, also induced a significant increase in PAI-1 mRNA levels. These results are consistent with a report (43) that the rat PAI-1 promoter contains an active HIF-1alpha -responsive element that was critical for the induction of PAI-1 by hypoxia. Although HREs have not been previously reported in the human PAI-1 promoter, sequences matching the consensus site for this binding element are located in the human PAI-1 promoter region. Transiently transfected plasmids expressing the full-length PAI-1 promoter linked to luciferase were significantly induced by nickel (data not shown). Further studies are needed to demonstrate which potential HREs or other transcription factor binding sites are necessary and sufficient for the induction by nickel.

In conclusion, the results of this study demonstrate that nickel transcriptionally activates PAI-1 by a mechanism that involves increases in HIF-1alpha protein mediated by ERK but does not require the NADPH oxidase complex, the mitochondrial electron transport chain, or increases in reactive oxygen species. Further investigation is needed to identify the rate-limiting upstream signaling pathways involved in nickel-induced stabilization of HIF-1alpha and the enhanced transcriptional activation of profibrotic genes such as PAI-1. These investigations will provide a better understanding of the pathological mechanisms of pulmonary fibrosis, cancer, and other diseases associated with exposure to nickel.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Environmental Health Sciences Grant ES-07373; National Heart, Lung, and Blood Institute Grant HL-52738; and National Institute of Diabetes and Digestive and Kidney Diseases Grant T32-DK-07301-22 (to A. S. Andrew).


    FOOTNOTES

Address for reprint requests and other correspondence: A. Barchowsky, Dept. of Pharmacology and Toxicology, Dartmouth Medical School, 7650 Remsen, Hanover, NH 03755-3835 (E-mail: barchowsky{at}dartmouth.edu).

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.

Received 15 December 2000; accepted in final form 9 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Acker, H, Dufau E, Huber J, and Sylvester D. Indications to an NADPH oxidase as a possible pO2 sensor in the rat carotid body. FEBS Lett 256: 75-78, 1989[ISI][Medline].

2.   Agani, F, and Semenza GL. Mersalyl is a novel inducer of vascular endothelial growth factor gene expression and hypoxia-inducible factor 1 activity. Mol Pharmacol 5: 749-754, 1998.

3.   Agani, FH, Pichiule P, Chavez JC, and LaManna JC. The role of mitochondria in the regulation of hypoxia-inducible factor 1 expression during hypoxia. J Biol Chem 275: 35863-35867, 2000[Abstract/Free Full Text].

4.  Agency for Toxic Substances and Disease Registry. (1997). Nickel. [Online]. ATSDR. http://www.atsdr.cdc.gov/toxfaq.html [1999, April 14].

5.   Andrew, A, and Barchowsky A. Nickel-induced plasminogen activator inhibitor-1 expression inhibits the fibrinolytic activity of human airway epithelial cells. Toxicol Appl Pharmacol 168: 50-57, 2000[ISI][Medline].

6.   Aragones, J, Jones DR, Martin S, San Juan MA, Alfranca A, Vidal F, Vara A, Merida I, and Landazuri MO. Evidence for the involvement of diacylglycerol kinase in the activation of hypoxia-inducible transcription factor-1 by low oxygen tension. J Biol Chem 276: 10548-10555, 2001[Abstract/Free Full Text].

7.   Barchowsky, A, Lannon BM, Elmore LC, and Treadwell MD. Increased focal adhesion kinase- and urokinase-type plasminogen activator receptor-associated cell signaling in endothelial cells exposed to asbestos. Environ Health Perspect 105: 1131-1137, 1997[ISI][Medline].

8.   Barchowsky, A, Roussel RR, Krieser RJ, Mossman BT, and Treadwell MD. Expression and activity of urokinase and its receptor in endothelial and pulmonary epithelial cells exposed to asbestos. Toxicol Appl Pharmacol 152: 388-396, 1998[ISI][Medline].

9.   Benson, JM, Carpenter RL, Hahn FF, Haley PJ, Hanson RL, Hobbs CH, Pickrell JA, and Dunnick JK. Comparative inhalation toxicity of nickel subsulfide to F344/N rats and B6C3F1 mice exposed for 12 days. Fundam Appl Toxicol 9: 251-265, 1987[ISI][Medline].

10.   Berra, E, Milanini J, Richard DE, Le Gall M, Vinals F, Gothie E, Roux D, Pages G, and Pouyssegur J. Signaling angiogenesis via p42/p44 MAP kinase and hypoxia. Biochem Pharmacol 60: 1171-1178, 2000[ISI][Medline].

11.   Bunn, HF, Gu J, Huang LE, Park JW, and Zhu H. Erythropoietin: a model system for studying oxygen-dependent gene regulation. J Exp Biol 201: 1197-1201, 1998[Abstract/Free Full Text].

12.   Caniggia, I, Mostachfi H, Winter J, Gassmann M, Lye SJ, Kuliszewski M, and Post M. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFbeta(3). J Clin Invest 105: 577-587, 2000[Abstract/Free Full Text].

13.   Chandel, NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, and Schumacker PT. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci USA 95: 11715-11720, 1998[Abstract/Free Full Text].

14.   Chandel, NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, and Schumacker PT. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem 275: 25130-25138, 2000[Abstract/Free Full Text].

15.   Chen, CY, Huang YL, and Lin TH. Association between oxidative stress and cytokine production in nickel-treated rats. Arch Biochem Biophys 356: 127-132, 1998[ISI][Medline].

16.   Clerk, A, Harrison JG, Long CS, and Sugden PH. Pro-inflammatory cytokines stimulate mitogen-activated protein kinase subfamilies, increase phosphorylation of c-Jun and ATF2 and upregulate c-Jun protein in neonatal rat ventricular myocytes. J Mol Cell Cardiol 31: 2087-2099, 1999[ISI][Medline].

17.   Coogan, TP, Latta DM, Snow ET, and Costa M. Toxicity and carcinogenicity of nickel compounds. Crit Rev Toxicol 19: 341-384, 1989[Medline].

18.   Costa, M, Simmons-Hansen J, Bedrossian CW, Bonura J, and Caprioli RM. Phagocytosis, cellular distribution, and carcinogenic activity of particulate nickel compounds in tissue culture. Cancer Res 41: 2868-2876, 1981[Abstract].

19.   Cotgreave, IA. N-acetylcysteine: pharmacological considerations and experimental and clinical applications. Adv Pharmacol 38: 205-227, 1997[Medline].

20.   D'Angelo, G, Ladoux A, and Frelin C. Hypoxia-induced transcriptional activation of vascular endothelial growth factor is inhibited by serum. Biochem Biophys Res Commun 267: 334-338, 2000[ISI][Medline].

21.   Ebert, BL, and Bunn HF. Regulation of transcription by hypoxia requires a multiprotein complex that includes hypoxia-inducible factor 1, an adjacent transcription factor, and p300/CREB binding protein. Mol Cell Biol 18: 4089-4096, 1998[Abstract/Free Full Text].

22.   Fandrey, J, Frede S, and Jelkmann W. Role of hydrogen peroxide in hypoxia-induced erythropoietin production. Biochem J 303: 507-510, 1994[ISI][Medline].

23.   Gleadle, JM, Ebert BL, and Ratcliffe PJ. Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing. Eur J Biochem 234: 92-99, 1995[Abstract].

24.   Goebeler, M, Roth J, Brocker EB, Sorg C, and Schulze-Osthoff K. Activation of nuclear factor-kappa B and gene expression in human endothelial cells by the common haptens nickel and cobalt. J Immunol 155: 2459-2467, 1995[Abstract].

25.   Goldberg, MA, Dunning SP, and Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 242: 1412-1415, 1988[ISI][Medline].

26.   Gong, W, Hao B, Mansy SS, Gonzalez G, Gilles-Gonzalez MA, and Chan MK. Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction. Proc Natl Acad Sci USA 95: 15177-15182, 1998[Abstract/Free Full Text].

27.   Gu, YZ, Hogenesch JB, and Bradfield CA. The PAS superfamily: sensors of environmental and developmental signals. Annu Rev Pharmacol Toxicol 40: 519-611, 2000[ISI][Medline].

28.   Haber, LT, Allen BC, and Kimmel CA. Non-cancer risk assessment for nickel compounds: issues associated with dose-response modeling of inhalation and oral exposures. Toxicol Sci 43: 213-229, 1998[Abstract].

29.   Ho, VT, and Bunn HF. Effects of transition metals on the expression of the erythropoietin gene: further evidence that the oxygen sensor is a heme protein. Biochem Biophys Res Commun 223: 175-180, 1996[ISI][Medline].

30.   Huang, LE, Gu J, Schau M, and Bunn HF. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 95: 7987-7992, 1998[Abstract/Free Full Text].

31.   Huang, X, Frenkel K, Klein CB, and Costa M. Nickel induces increased oxidants in intact cultured mammalian cells as detected by dichlorofluorescein fluorescence. Toxicol Appl Pharmacol 120: 29-36, 1993[ISI][Medline].

32.   Huang, X, Klein CB, and Costa M. Crystalline Ni3S2 specifically enhances the formation of oxidants in the nuclei of CHO cells as detected by dichlorofluorescein. Carcinogenesis 15: 545-548, 1994[Abstract].

33.   Iwamae, S, Tsukagoshi H, Hisada T, Uno D, and Mori M. A possible involvement of oxidative lung injury in endotoxin-induced bronchial hyperresponsiveness to substance P in guinea pigs. Toxicol Appl Pharmacol 151: 245-253, 1998[ISI][Medline].

34.   Jiang, BH, Rue E, Wang GL, Roe R, and Semenza GL. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem 271: 17771-17778, 1996[Abstract/Free Full Text].

35.   Jones, JG, and Warner CG. Chronic exposure to iron oxide, chromium oxide, and nickel oxide fumes of metal dressers in a steelworks. Br J Ind Med 29: 169-177, 1972[ISI][Medline].

36.   Margerum, DW, and Anliker SL. Nickel(III) chemistry and properties of the peptide complexes of Ni(II) and Ni(III). In: The Bioinorganic Chemistry of Nickel, edited by Lancaster JR, Jr.. New York: VCH Publishers, 1988, p. 29-51.

37.   Minet, E, Arnould T, Michel G, Roland I, Mottet D, Raes M, Remacle J, and Michiels C. ERK activation upon hypoxia: involvement in HIF-1 activation. FEBS Lett 468: 53-58, 2000[ISI][Medline].

38.   Namiki, A, Brogi E, Kearney M, Kim EA, Wu T, Couffinhal T, Varticovski L, and Isner JM. Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells. J Biol Chem 270: 31189-31195, 1995[Abstract/Free Full Text].

39.   Norris, ML, and Millhorn DE. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene. J Biol Chem 270: 23774-23779, 1995[Abstract/Free Full Text].

40.   O'Donnell, BV, Tew DG, Jones OT, and England PJ. Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem J 290: 41-49, 1993[ISI][Medline].

41.   Oller, AR, Costa M, and Oberdorster G. Carcinogenicity assessment of selected nickel compounds. Toxicol Appl Pharmacol 143: 152-166, 1997[ISI][Medline].

42.   Patierno, SR, Sugiyama M, Basilion JP, and Costa M. Preferential DNA-protein cross-linking by NiCl2 in magnesium-insoluble regions of fractionated Chinese hamster ovary cell chromatin. Cancer Res 45: 5787-5794, 1985[Abstract].

43.   Pinsky, DJ, Liao H, Lawson CA, Yan SF, Chen J, Carmeliet P, Loskutoff DJ, and Stern DM. Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition. J Clin Invest 102: 919-928, 1998[Abstract/Free Full Text].

44.   Porwol, T, Ehleben W, Zierold K, Fandrey J, and Acker H. The influence of nickel and cobalt on putative members of the oxygen-sensing pathway of erythropoietin-producing HepG2 cells. Eur J Biochem 256: 16-23, 1998[Abstract].

45.   Richard, DE, Berra E, Gothie E, Roux D, and Pouyssegur J. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1alpha (HIF-1alpha) and enhance the transcriptional activity of HIF-1. J Biol Chem 274: 32631-32637, 1999[Abstract/Free Full Text].

46.   Rose, RC, and Bode AM. Biology of free radical scavengers: an evaluation of ascorbate. FASEB J 7: 1135-1142, 1993[Abstract/Free Full Text].

47.   Salnikow, K, Blagosklonny MV, Ryan H, Johnson R, and Costa M. Carcinogenic nickel induces genes involved with hypoxic stress. Cancer Res 60: 38-41, 2000[Abstract/Free Full Text].

48.   Salnikow, K, Gao M, Voitkun V, Huang X, and Costa M. Altered oxidative stress responses in nickel-resistant mammalian cells. Cancer Res 54: 6407-6412, 1994[Abstract].

49.   Salnikow, K, Su W, Blagosklonny MV, and Costa M. Carcinogenic metals induce hypoxia-inducible factor-stimulated transcription by reactive oxygen species-independent mechanism. Cancer Res 60: 3375-3378, 2000[Abstract/Free Full Text].

50.   Semenza, GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 15: 551-578, 1999[ISI][Medline].

51.   Semenza, GL. Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochem Pharmacol 59: 47-53, 2000[ISI][Medline].

52.   Semenza, GL, Roth PH, Fang HM, and Wang GL. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem 269: 23757-23763, 1994[Abstract/Free Full Text].

53.   Sen, P, and Costa M. Pathway of nickel uptake influences its interaction with heterochromatic DNA. Toxicol Appl Pharmacol 84: 278-285, 1986[ISI][Medline].

54.   Stolk, J, Hiltermann TJ, Dijkman JH, and Verhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J Respir Cell Mol Biol 11: 95-102, 1994[Abstract].

55.   Toya, T, Serita F, Sawatari K, and Fukuda K. Lung lesions induced by intratracheal instillation of nickel fumes and nickeloxide powder in rats. Ind Health 35: 69-77, 1997[ISI][Medline].

56.   Zhang, Q, Kusaka Y, Sato K, Nakakuki K, Kohyama N, and Donaldson K. Differences in the extent of inflammation caused by intratracheal exposure to three ultrafine metals: role of free radicals. J Toxicol Environ Health 53: 423-438, 1998[ISI].

57.   Zhou, J, Struthers AD, and Lyles GA. Differential effects of some cell signaling inhibitors upon nitric oxide synthase expression and nuclear factor-kappa B activation induced by lipopolysaccharide in rat aortic smooth muscle cells. Pharmacol Res 39: 363-373, 1999[ISI][Medline].

58.   Zundel, W, Schindler C, Haas-Kogan D, Koong A, Kaper F, Chen E, Gottschalk AR, Ryan HE, Johnson RS, Jefferson AB, Stokoe D, and Giaccia AJ. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev 14: 391-396, 2000[Abstract/Free Full Text].


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