Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755
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ABSTRACT |
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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)-1 signaling pathway. The involvement
of the NADPH oxidase complex, reactive oxygen species, and kinases in
mediating nickel-induced HIF-1
signaling was also investigated.
Addition of nickel to BEAS-2B human airway epithelial cells increased
HIF-1
protein levels and elevated PAI-1 mRNA levels. Pretreatment of
cells with the extracellular signal-regulated kinase inhibitor U-0126
partially blocked HIF-1
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-1
mRNA abolished nickel-stimulated increases
in PAI-1 mRNA. These data indicate that signaling through extracellular signal-regulated kinase and HIF-1
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
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INTRODUCTION |
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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)-1
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-1 and HIF-1
(27). In normal physiology, low cellular oxygen
concentrations increase HIF-1
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-1
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-1 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-1
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-1
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-1
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-1
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-1 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-1
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-1
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-1
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.
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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 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-1 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'), -actin, or HIF-1
(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
-actin by taking the ratio of the PAI-1 to
-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-1 or
-actin protein levels were
determined by Western blotting with a polyclonal antibody to HIF-1
(Transduction Laboratories, Lexington, KY) or a monoclonal antibody to
-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-1 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.
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RESULTS |
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Time course and dose response of nickel subsulfide-induced HIF-1
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-1
to induce transcription of the PAI-1
gene, we first examined the time course for nickel induction of
HIF-1
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-1
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-1
protein at the 8-h time point. In contrast, HIF-1
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-1
protein levels induced by nickel.
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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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Antioxidants, NADPH oxidase, and mitochondrial inhibitors do not
block nickel-induced HIF-1 protein.
Reactive oxygen species, the mitochondrial electron transport chain,
and the NADPH oxidase complex are potentially involved in mechanisms
that stabilize HIF-1
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-1
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-1
immunoblot in Fig. 3A shows that neither the antioxidant NAC
nor the mitochondrial inhibitor rotenone blocked nickel-induced
HIF-1
protein. Although the apocynin treatment decreased the
intensity of the nickel-induced HIF-1
band, the concomitant decrease
in basal HIF-1
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-1
protein by nickel. In fact, NAC seemed to have a stimulatory
effect on HIF-1
expression. The HIF-1
immunoblot in Fig.
3C confirms that pretreatment with the NADPH oxidase
inhibitors apocynin and DPI did not block HIF-1
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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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-1)
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-1
responses, induced a
significant increase in PAI-1 mRNA levels.
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HIF-1 antisense oligonucleotide blocks nickel-induced HIF-1
protein.
HIF-1
antisense oligonucleotide was used to investigate the
hypothesis that HIF-1
is necessary for PAI-1 transcriptional activation by nickel. To verify the effectiveness of the HIF-1
antisense oligonucleotide, it was added for up to 56 h followed by
a 6-h treatment with nickel subsulfide. HIF-1
sense oligonucleotide and PMA were added for 24 h as a control. The immunoblot in Fig. 5 shows that the HIF-1
antisense
oligonucleotide effectively inhibited nickel-induced HIF-1
protein
levels at all time points tested. The HIF-1
sense oligonucleotide
slightly elevated HIF-1
protein levels in these cells.
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HIF-1 antisense but not sense oligonucleotide blocks
nickel-induced PAI-1 mRNA.
To determine whether inhibiting HIF-1
nickel responsiveness with an
antisense oligonucleotide would inhibit induction of the endogenous
PAI-1 gene, cells were pretreated with HIF-1
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-1
sense
oligonucleotide had no effect on the induction of PAI-1 by nickel (Fig.
6, C and D).
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Upstream kinases may be involved in the stabilization of HIF-1
by nickel.
Reports in the literature (6, 14, 58) suggest that
phosphorylation of HIF-1
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-1
by nickel. The ERK inhibitor U-0126 partially
inhibited nickel-stimulated HIF-1
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-1
protein levels.
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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.
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DISCUSSION |
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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-1. The mechanisms for stimulating increases in HIF-1
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-1
protein levels or expression of
PAI-1. Eliminating HIF-1
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-1
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-1 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-
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-1
protein, one would expect that the addition of antioxidants would prevent nickel from increasing HIF-1
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-1
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-1
-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-1
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-1
(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-1 protein in response to hypoxia, it
does not affect HIF-1
expression induced by cobalt chloride or
deferoxamine (3, 13). In the present study, pretreatment with rotenone failed to inhibit the HIF-1
response to nickel (Fig.
3A), indicating that mitochondrial electron transport is probably not involved in the induction of HIF-1
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-1
protein stabilization (1, 23). If the NADPH oxidase complex were critical to the induction of HIF-1
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-1
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-1
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-1
(Fig. 3C).
Another possible mechanism for the action of nickel and the inability
of NAC to protect against increases in HIF-1 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-1
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-1 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-1
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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The involvement of PI3K and signaling through DAGK to increase
phosphatidic acid (6) have also been reported to mediate stabilization of HIF-1 in response to hypoxia. As shown in Fig. 7,
treatment with the DAGKI R-59949 partially inhibited stabilization of
HIF-1
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-1
and the induction of
PAI-1 mRNA by nickel.
The hypothesis that increased levels of the transcription factor
HIF-1 could be mediating nickel-induced transcription of the PAI-1
gene was supported by the data in Fig. 1 indicating that HIF-1
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-1
and PAI levels detectable at 24 h
(5). HIF-1
antisense oligonucleotide dramatically inhibited the induction of PAI-1 mRNA levels by nickel (Fig. 6). These
data implicate HIF-1
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-1
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-1
-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-1 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-1
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.
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ACKNOWLEDGEMENTS |
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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 Grant T32-DK-07301-22 (to A. S. Andrew).
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FOOTNOTES |
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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.
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