AP-1-dependent induction of plasminogen activator inhibitor-1 by nickel does not require reactive oxygen

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

Inhalation of nickel dust has been associated with an increased incidence of pulmonary fibrosis. Nickel may promote fibrosis by transcriptionally activating plasminogen activator inhibitor (PAI)-1 and inhibiting fibrinolysis. The current studies examined whether nickel stimulated the PAI-1 promoter though an oxidant-sensitive activator protein (AP)-1 signaling pathway. Addition of nickel to BEAS-2B human airway epithelial cells stimulated intracellular oxidation, induced c-Jun and c-Fos mRNA levels, increased phospho- and total c-Jun protein levels, and elevated PAI-1 mRNA levels over a 24-h time course. Pretreatment of the cells with antioxidants did not affect increased c-Jun protein or PAI-1 mRNA levels. Expression of the dominant negative inhibitor of AP-1, TAM67, prevented nickel-stimulated AP-1 DNA binding, AP-1-luciferase reporter construct activity, and PAI-1 mRNA levels. Overexpression of c-Jun, however, failed to induce the AP-1 luciferase reporter construct or PAI-1 mRNA levels. These data indicated that nickel activated AP-1 through an oxidant-independent pathway and that basal AP-1 is necessary for nickel-induced expression of PAI-1.

nickel subsulfide; activator protein-1; reactive oxygen species; BEAS-2B cells; hypoxia-inducible factor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EXPOSURE TO NICKEL BY INHALATION has been associated with an increased incidence of respiratory diseases such as pulmonary fibrosis (6), acute respiratory distress syndrome (13), and lung cancer (22). It is hypothesized that certain metals present in airborne particulate matter, including nickel, may be responsible for the generation of free radicals, contributing to the respiratory diseases associated with the inhalation of these particles (19, 26, 36). A potential mechanism through which inhaled particles can influence fibrosis is inactivation of the pulmonary fibrinolytic cascade. Previous research by this laboratory (2) demonstrated that noncytotoxic levels of particulate nickel subsulfide inhibit fibrinolysis by airway epithelial cells though transcriptional induction of plasminogen activator inhibitor (PAI)-1. However, the signaling mechanisms for this gene induction by nickel are unknown.

Nickel subsulfide is one of the more harmful forms of nickel. It is durable enough to be retained in the lung, which contrasts with soluble nickel that is rapidly cleared. Macrophages and epithelial cells ingest particles of nickel subsulfide into endocytic vesicles that fuse with acidic lysosomes (1). This acidification releases Ni2+, which reacts with cytoplasmic proteins (22) or heterochromatic regions of DNA (24, 32). The bound metal can then redox cycle to produce reactive oxygen species or affect protein conformations (18, 22).

Several investigators (15, 16) have noted increased production of reactive oxygen intermediates after nickel exposure. Ultrafine nickel particles have also been shown to generate substantial free radical activity as measured by depletion of supercoiled plasmid DNA (37). The generation of free radicals by nickel has been cited as a mechanism for the induction of transcription factors such as nuclear factor-kappa B and genes such as intercellular adhesion molecule-1 (12) and interleukin-1 (9). These data support the hypothesis that the increases in reactive oxygen species generated by nickel could be part of the signaling cascades that lead to transcriptional activation of PAI-1 by nickel.

The PAI-1 promoter contains several active transcription factor binding sites that mediate induction in response to a variety of agents. These binding sites include activator protein (AP)-1 (17), transcription factor µE3 (TFE3), and Smad binding elements (14) that promote PAI-1 induction by transforming growth factor (TGF)-beta . In addition, hypoxia-induced PAI-1 expression in the rat requires a putative hypoxia-responsive element, hypoxia-inducible factor (HIF) (25). AP-1 is a family of transcription factor binding sites that bind Jun-Jun homodimers as well as Jun-Fos heterodimeric transcription factor complexes (4). Transcriptional competence of bound c-Jun occurs after phosphorylation on serines 73 and 63 by c-Jun NH2-terminal kinase and other mitogen-activated protein kinases. AP-1 activation is involved in the induction of PAI-1 by fibrin fragments (23) and TGF-beta (17). Redox-sensitive signaling mediates AP-1 activation in response to treatment with other agents including H2O2 (28), serum, thrombin (27), metals (31), and particulate matter (20).

The goal of this study was to test the hypothesis that nickel increases the redox status of the cell, leading to activation of the transcription factor AP-1 in the promoter of the PAI-1 gene. The results of this investigation indicate that nickel increases both the intracellular oxidation state and AP-1 transactivation. Some basal AP-1 is critical to the ability of nickel to stimulate PAI-1 mRNA levels; however, reactive oxygen species are not required for this response. This lack of a need for redox signaling may relate to the prolonged time course for AP-1 activation, which differs from traditional immediate-early gene activation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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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 baked at 200°C for 18 h. This sterile 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 in clonogenic assays, the addition of 2.34 µg Ni/cm2 of nickel subsulfide is not toxic to this cell model (2). In the present study, the cells were treated for up to 24 h with noncytotoxic doses of nickel subsulfide (0.58-2.34 µg Ni/cm2, prepared as described in Nickel) (2). Phorbol 12-myristate 13-acetate (PMA; 2-100 nM) was used as a positive control. The cells were pretreated with 2-10 mM N-acetyl-L-cysteine (NAC), 2 mM ascorbic acid, or 2 mM superoxide dismutase for 30 min. All chemicals were purchased from Sigma unless otherwise mentioned. HIF-1alpha phosphorothioate antisense and sense oligonucleotides were synthesized in the Molecular Biology Core at Dartmouth University (Hanover, NH) according to sequences published by Caniggia et al. (8). The cells were incubated for 24 h with 10 µM sense or antisense oligonucleotide before nickel treatment. Treatment with a HIF-1alpha antisense oligonucleotide caused a sevenfold decrease in nickel-induced HIF-1alpha protein levels for a period of at least 48 h as determined by Western blot (3).

mRNA levels. Total cellular RNA was harvested with TRIzol reagent (GIBCO BRL, Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions and quantified by spectrophotometric absorbance at 260 nm. Reverse transcription-polymerase chain reaction (RT-PCR) was performed as described previously (2, 33, 34) with PAI-1 (forward, 5'-cgcctcttccacaaatcag-3' and reverse, 5'-atgcgggctgagactatga-3'), beta -actin (33), c-Jun (forward, 5'-ttaacagtgggtgccaactcatgctaacgc-3' and reverse, 5'-gagtcgaatgttaggtccatgcagttcttg-3'), or c-Fos (forward, 5'-cctgtcaagagcatcagcagcatgg-3' and reverse, 5'-gagtacaggtgaccaccggagtgc-3') specific primers that were synthesized in the Molecular Biology Core at Dartmouth University 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. The PAI-1 mRNA expression was normalized to the housekeeping gene beta -actin by taking the ratio of PAI-1 to beta -actin band density.

Protein levels. The effects of nickel on phosphorylated or total c-Jun, Jun B, Jun D, and HIF-1alpha protein levels were determined by Western blotting with polyclonal antibodies to phospho-c-Jun (New England Biolabs, Beverly, MA) and the DNA binding domains of c-Jun/AP-1, Jun B, Jun D (Santa Cruz Biotechnology, Santa Cruz, CA) and HIF-1alpha (Transduction Laboratories, Lexington, KY) or with a monoclonal antibody to beta -actin (Sigma). Immunoblotting was essentially as previously described (2, 33). 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 (c-Jun) or 24 h at 4°C (phospho-c-Jun). Secondary sheep anti-mouse IgG or donkey anti-rabbit IgG antibody 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).

Transfections. Seventy percent confluent BEAS-2B cells grown in 12-well plates were transfected with 2.8 µl of LipofectAMINE and 4.2 µl of PLUS reagent (GIBCO BRL) in 300 µl LHC-9 medium/well according to the manufacturer's protocol. The cells were incubated with transfection reagents [0.5 µg of AP-1 luciferase plasmid (Clontech, Palo Alto, CA) or 1 µg of cytomegalovirus, c-Jun, or TAM67 (obtained from Dr. Michael Birrer, National Institutes of Health, Rockville, MD)] and 0.2 µg of enhanced green fluorescent protein (GFP) plasmid for 3 h. After the transfection period, the volume of LHC-9 medium in each well was increased to 1 ml, and the cells were allowed to recover overnight before treatment. After treatment, the cells were washed three times with PBS, and the transfection efficiency in each well was assessed by measuring GFP fluorescence with a fluorescent plate reader at 485-nm excitation and 508-nm emission. Transfection efficiency of BEAS-2B cells with this optimized protocol was estimated from GFP expression to be ~20-30%.

Luciferase assay. Cells were harvested in 80 µl of lysis buffer (25 mmol/l of glycylglycine, 4 mmol/l of EGTA, 15 mmol/l of MgSO4, 1% Triton X-100, and 1 mmol/l of dithiothreitol). The lysates were then centrifuged at 13,000 g at 4°C for 5 min. Luciferase activity was determined with 50 µl of supernatant and 150 µl of fresh assay buffer (25 mmol/l of glycylglycine, 15 mmol/l of potassium phosphate, 15 mmol/l of MgSO4, 4 mmol/l of EDTA, 2 mmol/l of ATP, and 1 mmol/l of dithiothreitol) in a luminometer by adding 50 µl of 400 µmol/l of luciferin.

Electrophoretic mobility shift assays. Electrophoretic mobility shift assays (EMSAs) were performed essentially as described (34). Briefly, after experimental exposure, the cells were rinsed twice with PBS and nuclear extracts were prepared. Equal amounts of nuclear protein (5 µg) were incubated for 20 min at room temperature with a 5' 32P-labeled double-strand oligonucleotide that contained a consensus AP-1 binding sequence (as underlined; 5'-cggcttgatgactcagccggaa-3'; Santa Cruz Biotechnology). This sequence is identical to an AP-1 element in the human PAI-1 promoter. The incubation mixtures were separated by 4% nondenaturing PAGE under high ionic conditions, and the bands were detected by autoradiography.

Statistics. Statistical analysis was performed on data pooled from separate experiments performed in duplicate or triplicate 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.


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ABSTRACT
INTRODUCTION
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Nickel induction of PAI-1 mRNA is not blocked by antioxidants. Data obtained in this (3) and other (15, 16, 37) laboratories indicate that nickel promotes redox cycling as indicated by increased intracellular oxidation of 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein diacetate (CM-DCFH-DA). Pretreatment with the antioxidants NAC and ascorbic acid blocked the nickel-induced increases in reactive oxygen species (3). The higher intracellular oxidation state indicated by the increased CM-DCFH-DA fluorescence after nickel exposure suggested that reactive oxygen species were required for mediating the induction of PAI-1 by nickel. To test this hypothesis, cells were pretreated with the antioxidants NAC, ascorbic acid, or superoxide dismutase followed by exposure to nickel subsulfide. After exposure to nickel, total RNA was extracted from the cells and PAI-1 mRNA levels were determined. RT-PCR analysis in Fig. 1 demonstrates that PAI-1 mRNA induction by nickel cannot be blocked by pretreatment with antioxidants.


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Fig. 1.   Nickel induction of plasminogen activator inhibitor (PAI)-1 mRNA is not blocked by antioxidants. A: pretreatment of cells with 300 U/ml of superoxide dismutase (SOD) or 2 mM ascorbic acid for 30 min or with 2 or 10 mM N-acetyl-L-cysteine (NAC) for 18 h was followed by 24 h of incubation in the absence (-) and presence (+) of nickel subsulfide (Ni3S2; 2.34 µg Ni/cm2). Total RNA was collected, and PAI-1 and beta -actin mRNA levels were determined by RT-PCR followed by staining with ethidium bromide. B: PCR products, including those in A, were stained with PicoGreen double-strand DNA stain. Data are means ± SD of ratios of PAI-1 to beta -actin fluorescence after PicoGreen staining from at least 12 replicates; n = 6 experiments. *** P < 0.001 vs. respective control.

Nickel induces c-Jun and c-Fos mRNA. AP-1 is necessary for transcriptional activation of PAI-1 expression by profibrotic factors such as fibrin fragments and TGF-beta (17, 23). Therefore, the time course for the effects of nickel on the expression of the classic AP-1 binding partners c-Jun and c-Fos was examined. The data in Fig. 2 demonstrate that nickel induces a relatively slow but sustained elevation in mRNA levels for both factors. Basal levels of c-Jun mRNA were higher than for c-Fos mRNA and had a more transient elevation. Significant induction c-Jun mRNA occurred by 8 h and peaked by 24 h. In contrast, c-Fos mRNA levels were significantly increased by 4 h, reached a maximum by 24 h, and were still elevated at 48 h. These increases are consistent with the time course for the induction of PAI-1 mRNA and protein levels by nickel (2).


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Fig. 2.   Nickel induces c-Jun and c-Fos mRNAs. A: total RNA was collected from cells exposed to 2.34 µg Ni/cm2 for indicated times. RT-PCR followed by staining with ethidium bromide was used to determine c-Jun, c-Fos, and beta -actin mRNA levels. B and C: ratios of c-Jun and c-Fos, respectively, to beta -actin density of ethidium bromide-stained bands of PCR product in ultraviolet-transluminated 2% agarose gels. ctrl, Control. Values are mean ± SD; n = 3 experiments. Significantly different from control value: * P < 0.05; ** P < 0.01; *** P < 0.001.

Nickel induces phospho- and total c-Jun protein. To examine whether the increased c-Jun mRNA levels translated into increased protein expression, the effects of nickel on the protein levels of c-Jun and other Jun family members were determined. In Fig. 3, cells were treated with nickel subsulfide for up to 24 h. The immunoblot in Fig. 3A indicated that total c-Jun protein levels increased after 8 h of treatment with nickel subsulfide and continued to rise at 24 h. Immunoblotting for c-Jun phosphorylated on serine-73 (Fig. 3B) indicated that nickel increased phospho-c-Jun levels beginning at 4 h and followed the increase in c-Jun protein over the 24-h period. Preincubation with NAC does not inhibit nickel-stimulated total or phospho-c-Jun protein levels (Fig. 3C), indicating that oxidants are not proximal to the induced protein expression. Expression of other Jun family members, such as Jun B and Jun D, are not induced at the 24-h time point (Fig. 3D). In contrast, Jun B protein levels decrease transiently 2-8 h after nickel treatment.


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Fig. 3.   Nickel induces phospho- and total c-Jun protein. A: confluent cells were exposed to 2.34 µg Ni/cm2 for 8 or 24 h or 0.58 µg Ni/cm2 for 24 h. Total protein was harvested in 2× SDS buffer. After PAGE separation and transfer to polyvinylidene difluoride membranes, Western analysis was performed with an antibody for total c-Jun, and enhanced chemiluminescence was performed for detection of bound secondary antibody. B: cells were exposed for indicated times to varying amounts of nickel subsulfide. Proteins were harvested and processed as in A and Western analysis was performed with an antibody to phospho (p)-c-Jun (ser 73). C: untreated cells or cells pretreated for 18 h with NAC were exposed to 2.34 µg Ni/cm2 for 24 h. Western blots were performed with antibody to total c-Jun or phospho-c-Jun. D: cells were exposed to 2.34 µg Ni/cm2 for indicated times. Western analysis was performed with antibodies to Jun B or Jun D. All data are representative of 3 replicate experiments.

The AP-1 inhibitor TAM67 blocks nickel-induced AP-1 transactivation. To determine whether nickel induces AP-1-driven transcription, BEAS-2B cells were transiently transfected with an empty plasmid vector or with an AP-1-driven luciferase reporter construct in the absence and presence of plasmids expressing either c-Jun or the dominant negative c-Jun mutant TAM67. Western analysis with an antibody to the COOH terminus of c-Jun showed that maximal c-Jun or TAM67 protein expression was obtained 8-24 h after transfection (Fig. 4A). TAM67 protein appears as a lower band on the blot because the amino-terminal 67 amino acids containing the transactivation domain were deleted (7). In accordance with increased c-Jun and c-Fos expression, 24-h exposure to either nickel or PMA significantly increased AP-1-driven luciferase activity (Fig. 4B). Cotransfection of the AP-1 reporter construct with any of the c-Jun constructs decreased basal AP-1 activity. More importantly, there was no stimulation of AP-1 by nickel or PMA in cells cotransfected with TAM67 (Fig. 4B). It is also important to note that overexpression of c-Jun alone did not activate AP-1-dependent transcription in this model (Fig. 4B).


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Fig. 4.   Activator protein (AP)-1 inhibitor TAM67 blocks transcription of an AP-1-driven luciferase construct induced by nickel. A: cells grown in 12-well plates were transfected with 0.5 µg/well of cytomegalovirus (CMV) vector, TAM67, or c-Jun plasmid for 3 h. The next day, cells were treated for 2, 8, or 24 h with 2.34 µg Ni/cm2. Total protein was harvested in 2× SDS buffer. and Western blots were performed with an antibody to the COOH-terminal fragment of total c-Jun. B: cells were transfected with 0.5 µg/well of AP-1 luciferase construct or the control construct pTAL and 0.2 µg/well of enhanced green fluorescent protein (GFP) in the presence and absence of 1 µg/well of CMV vector, TAM67, or c-Jun plasmid. After 24 h of equilibration, cells were left untreated or were incubated with nickel subsulfide (2.34 µg Ni/cm2) or phorbol 12-myristate 13-acetate (PMA; 2 nM) for 24 h. After treatment, luciferase activity was assayed. RLU, relative light units. Data are means ± SD of ratios of AP-1 or pTAL luciferase activity to GFP fluorescence; n = 6 experiments. Significantly different from control value: * P < 0.05; *** P < 0.001.

The AP-1 inhibitor TAM67 blocks nickel-induced PAI-1 mRNA. Transient transfection of the BEAS-2B cells with TAM67 was used to test the hypothesis that the transcription factor AP-1 was required for nickel-induced PAI-1 mRNA. Cells were transfected with an empty vector or with plasmids that overexpress either c-Jun or TAM67. After a 24-h period to allow full expression of the proteins, nickel or PMA was added to induce PAI-1 transcription. RT-PCR analysis of PAI-1 mRNA demonstrated that TAM67 blocked nickel-induced PAI-1 mRNA levels (Fig. 5). These data indicate that AP-1 is necessary for transcriptional activation of PAI-1 in response to nickel.


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Fig. 5.   AP-1 inhibitor TAM67 blocks nickel-induced PAI-1 mRNA. A: cells were transfected with 1 µg/well of CMV vector, TAM67, or c-Jun plasmid for 3 h. The next day, cells were left untreated or were exposed for 24 h to 2.34 µg Ni/cm2 or 2 nM PMA. Total RNA was harvested, and RT-PCR was performed with primers for PAI-1 or beta -actin mRNA. Bands of PCR products were ethidium bromide-stained in ultraviolet-transluminated 2% agarose gels. B: PCR products, including those in A, were stained with PicoGreen double-strand DNA stain. Data are means ± SD of ratios of fluorescence of PicoGreen bound to PAI-1 to beta -actin DNA products from separate cultures; n = 6 experiments. Significantly different from control value: ** P < 0.01; *** P < 0.001.

TAM67 blocks AP-1 DNA binding. EMSA was used to determine the effects of nickel and the dominant negative AP-1 construct TAM67 on AP-1 DNA binding. Groups of cells were left untreated or were transfected with either the empty cytomegalovirus vector or TAM67. After recovery, the cells were incubated with and without nickel or PMA for 9 h. Nuclear proteins were isolated, and 5 µg were assayed by EMSA for DNA binding. The data in Fig. 6 demonstrate significant basal binding of AP-1 that obscures the effects of nickel and PMA. Transfection with either plasmid reduced basal binding, which is consistent with the decreased AP-1 transactivation seen in Fig. 4. Nickel slightly increased AP-1 binding in the vector-transfected cells. In contrast, TAM67 blocked both nickel- and PMA-stimulated DNA binding.


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Fig. 6.   TAM67 blocks AP-1 DNA binding. A: cells were transfected with 1 µg/well of the inhibitor TAM67 or the control vector CMV for 3 h. The next day, nuclear extracts were harvested from control cells or cells exposed to 2.34 µg Ni/cm2 or 10 nM PMA for 9 h. Electrophoretic mobility shift assay for AP-1 was performed as described in METHODS. B: data were obtained by densitometry from results in A. ** P < 0.01 vs. respective control.

Nickel activates HIF-1alpha and AP-1 through separate pathways. Another study by this laboratory (3) demonstrated that a HIF antisense cDNA oligonucleotide abolished nickel-induced PAI-1 expression. Therefore, transfections with c-Jun and TAM67 were repeated to investigate the possibility that the HIF-1alpha response to nickel is dependent on AP-1. The immunoblot in Fig. 7A shows that blocking AP-1 with TAM67 had no effect on nickel-stimulated HIF-1alpha protein levels. The converse hypothesis that stabilization of HIF-1alpha is necessary for increased AP-1 activity was tested in HIF antisense oligonucleotide-treated cells. The data in Fig. 7B demonstrate that eliminating HIF-1alpha expression with an antisense oligonucleotide had little, if any, effect on nickel-induced c-Jun protein levels after nickel exposure. These data suggest that nickel activates the transcription factors AP-1 and HIF-1 through separate pathways.


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Fig. 7.   Nickel activates hypoxia-inducible factor (HIF)-1alpha and AP-1 through separate pathways. A: cells were transfected with 1 µg/well of the inhibitor TAM67 or the control vectors CMV or c-Jun for 3 h. The next day, cells were exposed to 2.34 µg Ni/cm2 or 2 nM PMA for 24 h. B: cells were pretreated with 10 µM HIF-1alpha antisense oligonucleotide for 24 h before 24-h 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. After treatment, total protein was harvested in 2× SDS buffer, and Western blots were performed with antibodies to HIF-1alpha and beta -actin (A) or c-Jun (B). Data are representative of at least 3 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of PAI-1 expression by nickel disrupts the fibrinolytic cascade in airway epithelial cells (2). A similar action in vivo would be expected to promote accumulation of fibrin and matrix proteins in the lung, a hallmark of pulmonary fibrosis. The cellular and molecular mechanisms through which nickel induces PAI-1 transcription are unknown. Therefore, these studies sought to distinguish between the possible signaling mechanisms leading to transactivation of the PAI-1 promoter, such as increased oxidant formation or AP-1 activation, to determine the requirements for induction by nickel.

Reactive oxygen species and redox activation of proteins are critical in mediating many cellular responses to environmental stimuli as varied as asbestos (20), airborne particulate matter (19, 36), and metals including nickel (9, 10, 30, 37). In addition, reactive oxygen species are often central to mechanisms for activating AP-1 (5, 20, 28, 35). Nickel increased the cellular oxidation state of BEAS-2B cells in the current model (3), which confirms observations made in other cell types (15, 16). Thus it was logical to hypothesize that nickel-induced reactive oxygen or redox activation could account for nickel-induced gene expression in these cells. However, the fact that antioxidants were effective in blocking nickel-induced increases in DCF fluorescence but were ineffective in blocking nickel-induced increases in c-Jun protein or PAI-1 mRNA (Figs. 1 and 3) indicates that reactive oxygen does not play a role in the transcriptional activation of AP-1 or PAI-1 by nickel.

The addition of nickel subsulfide to airway cells causes a sustained increase in PAI-1 mRNA and protein levels that suppresses fibrinolytic activity for >48 h (2). The mRNA levels continue to rise at 48 h, indicating either that nickel initiates a chronic change in the cell phenotype or that nickel subsulfide is slowly converted to an active species. The latter may explain why there is no burst of immediate-early gene response but a slow increase in the expression of AP-1 constituents. The prolonged time course of phenotypic change and increased inhibition of fibrinolysis in response to a metal particle that is known to persist in the lung, such as nickel subsulfide, could have profound effects on a progressive disorder like fibrosis.

Support for the necessity of the AP-1 pathway in transcriptional activation of the PAI-1 gene after exposure to nickel comes from inhibition of this activation by the truncated c-Jun expression plasmid TAM67. TAM67 is a dominant negative mutant of c-Jun that binds wild-type Jun or Fos family members but lacks the amino-terminal transactivation domain of c-Jun (7). Data in Figs. 4 and 5 demonstrate that nickel-induced AP-1 luciferase activity and PAI-1 mRNA are blocked by transfection with the TAM67 expression vector. Therefore, AP-1 appears to be necessary for nickel to induce transcriptional activation of PAI-1.

Nickel does not stimulate AP-1 activity through a traditional immediate-early gene response because it requires a prolonged time course for the expression of c-Jun and c-Fos (Figs. 2 and 3). However, this time course is consistent with the protracted time courses for both nickel-induced increases in PAI-1 protein levels and inhibition of fibrinolytic activity (2). The parallel increase in total and phosphorylated c-Jun suggests that the increase in total protein is more important for AP-1 activation than for nickel stimulation of upstream signaling cascades that phosphorylate c-Jun. Jun B and Jun D protein levels did not increase after nickel exposure and were unchanged at the 24-h time point (Fig. 3C). c-Jun is an efficient activator of AP-1-dependent gene expression, whereas Jun B and Jun D are not (4). Jun B can even act as a negative AP-1 regulator by competing with c-Jun and decreasing transactivation of AP-1 (4). Thus transient decreases in Jun B levels could aid activation of AP-1-driven genes by c-Jun. How this transient decrease in Jun B might contribute to nickel-induced PAI-1 expression is unclear because the time course precedes the time required for the onset of increased PAI-1 mRNA levels.

Although nickel induces c-Jun expression, overexpression of c-Jun alone is not sufficient to promote AP-1-driven luciferase activity (Fig. 4B) or increase PAI-1 mRNA levels (Fig. 5). As shown in Fig. 6, transfection with TAM67 blocked AP-1 DNA binding. These data may indicate that a basal level of c-Jun is necessary for the nickel responsiveness of PAI-1 but that increases in c-Fos or another transcription factor may be the primary factor driving PAI-1 transcriptional activation. Protein levels of c-Fos were not measured. However, the more rapid rise in c-Fos mRNA levels and their prolonged elevation relative to c-Jun mRNA (Fig. 2) suggest that this binding partner may be more important for PAI-1 expression in response to nickel. Finally, cooperation between AP-1 and stabilized HIF-1alpha may be required for full PAI-1 gene induction in response to nickel.

Nickel mimics hypoxia-induced gene activation by stabilizing HIF-1alpha protein (29). In a recent study, we (3) demonstrated that an antisense cDNA oligonucleotide that inhibits synthesis of HIF-1alpha effectively blocks nickel-induced PAI-1 expression. This observation raised the possibility that AP-1 might be mediating the HIF-1alpha response to nickel. Data in Fig. 7A, however, indicate that blocking AP-1 activity with TAM67 has no effect on the ability of nickel to increase HIF-1alpha protein levels. Although the transfection efficiency was <100%, transfection with TAM67 was sufficient to inhibit PAI-1 expression. Thus HIF-1alpha protein levels should decrease under these conditions if AP-1 were upstream from HIF-1alpha stabilization. Because inhibiting expression of HIF-1alpha protein with a HIF antisense oligonucleotide did not significantly block the ability of nickel to increase c-Jun protein (Fig. 7B), it appears that nickel probably activates AP-1 and HIF signaling via separate, parallel pathways.

Although activation of the AP-1 and HIF pathways appears to be separate, neither pathway is sufficient for nickel-induced PAI-1 expression. This lack of sufficiency is apparent from the ability of either TAM67 or HIF antisense oligonucleotide to completely abolish the BEAS-2B cell response to nickel. It is possible that the induction of PAI-1 by nickel involves cooperativity between HIF and AP-1. Cooperativity between these two transcription factors is required for hypoxia to induce several genes, including lactate dehydrogenase, erythropoietin, and tyrosine hydroxylase (11, 21). A multiprotein complex between adjacent transcription factors and the coactivator p300 mediates this cooperativity (11). Because the competent HIF elements of the human PAI-1 promoter have not been defined, further investigation is required to demonstrate that nickel exposure causes assembly of similar complexes to promote PAI-1 expression.

In conclusion, the data presented in this study support the hypothesis that AP-1 expression is required for nickel to transcriptionally activate the PAI-1 gene. Although nickel increases cellular oxidation, this increase did not appear to be involved in the activation of AP-1 or the induction of PAI-1. Nickel activates the transcription factors AP-1 and HIF-1alpha through separate pathways, but both factors are necessary for nickel-induced increases in PAI-1 mRNA. Further investigation of these nickel-activated signaling mechanisms and interactions between these transcription factors at the level of the PAI-1 promoter are needed to improve the understanding of the cellular and molecular pathways activated in response to nickel that inhibit fibrinolysis.


    ACKNOWLEDGEMENTS

We thank Dr. Michael Birrer (National Institutes of Health, Rockville, MD) for the TAM67 constructs.


    FOOTNOTES

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 T32-DK-07301.

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 1 December 2000; accepted in final form 9 April 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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