Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755
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ABSTRACT |
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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
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INTRODUCTION |
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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-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)-. 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-
(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.
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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-1 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-1
antisense
oligonucleotide caused a sevenfold decrease in nickel-induced HIF-1
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'), -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
-actin by taking the ratio of PAI-1 to
-actin
band density.
Protein levels.
The effects of nickel on phosphorylated or total c-Jun, Jun B, Jun D,
and HIF-1 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-1
(Transduction Laboratories,
Lexington, KY) or with a monoclonal antibody to
-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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RESULTS |
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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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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- (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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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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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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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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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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Nickel activates HIF-1 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-1
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-1
protein levels.
The converse hypothesis that stabilization of HIF-1
is necessary for
increased AP-1 activity was tested in HIF antisense
oligonucleotide-treated cells. The data in Fig. 7B
demonstrate that eliminating HIF-1
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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DISCUSSION |
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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-1 may
be required for full PAI-1 gene induction in response to nickel.
Nickel mimics hypoxia-induced gene activation by stabilizing HIF-1
protein (29). In a recent study, we (3)
demonstrated that an antisense cDNA oligonucleotide that inhibits
synthesis of HIF-1
effectively blocks nickel-induced PAI-1
expression. This observation raised the possibility that AP-1 might be
mediating the HIF-1
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-1
protein levels. Although
the transfection efficiency was <100%, transfection with TAM67 was
sufficient to inhibit PAI-1 expression. Thus HIF-1
protein levels
should decrease under these conditions if AP-1 were upstream from
HIF-1
stabilization. Because inhibiting expression of HIF-1
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-1 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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Birrer (National Institutes of Health, Rockville, MD) for the TAM67 constructs.
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FOOTNOTES |
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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.
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