Thoracic Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London SW3 6LY, United Kingdom
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ABSTRACT |
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Peroxynitrite, formed
by the reaction of nitric oxide (NO · ) with
superoxide anions (O0.25 mM, and this was increased with the
inclusion of SB-239063. Therefore, MAPKs may mediate signal
transduction pathways induced by peroxynitrite in lung epithelial cells
leading to cell death.
BEAS-2B cells; superoxide; nitric oxide; nitrosative stress; 3-morpholinosydnonimine
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INTRODUCTION |
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PEROXYNITRITE
(ONOO) is a potent oxidant generated by the rapid
nonenzymatic interaction between nitric oxide
(NO · ) and the superoxide anion radical
(O
causes oxidative damage to proteins, lipids, DNA,
and carbohydrates (6). One of the major protein
modifications induced by ONOO
is the nitration of
tyrosine residues on a variety of proteins to form the stable product
3-nitrotyrosine that may lead to a change of protein or enzyme function
(8, 11, 44). Moreover, 3-nitrotyrosine formation has been
found in the lungs, bronchoalveolar lavage fluid, and exhaled breath
taken from patients with acute respiratory distress syndrome and asthma
(16, 19, 25, 38, 41). However, the functional outcome of
ONOO
on human respiratory tract cells is so far unknown.
Recent evidence suggests that ONOO
can induce
apoptosis in pulmonary cells, but the mechanism of action has
not yet been identified (12).
The mitogen-activated protein kinase (MAPK) pathways are reported to be
involved in signaling pathways induced by NO · and O leading to the
modulation of gene expression.
The present study examined the effects of 3-morpholinosydnonimine
(SIN-1), a ONOO donor, on the proliferation of human
bronchial epithelial cells and the involvement of MAPKs in this process
to determine how ONOO
may play a role in inflammatory
airway diseases such as asthma and chronic obstructive pulmonary
disease (COPD).
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MATERIALS AND METHODS |
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Materials. The BEAS-2B cell line was purchased from American Type Culture Collection (Rockville, MD). Keratinocyte serum-free medium (K-SFM), bovine pituitary extract (BPE), and recombinant human epidermal growth factor (EGF) were purchased from GIBCO Life Technologies (Paisley, UK), SIN-1, Mn(III)tetrakis(N-methyl-4'-pyridyl)porphyrin (MnTMPyP), 2'-amino-3'-methoxyflavone (PD-98059), and cell counting kit-8 (CCK-8) were obtained from Alexis (Nottingham, UK). Antibodies against phosphorylated and total ERK, p38, and JNK were purchased from New England Biolabs (Hitchin, UK). Monoclonal antinitrotyrosine antibody was purchased from Calbiochem (La Jolla, CA). Enhanced chemiluminescence (ECL) reagent and Hybond-ECL nitrocellulose were obtained from Amersham (Little Chalfont, UK). 4-12% Bis-Tris SDS-PAGE gels and buffers were purchased from Novex (San Diego, CA). L-Buthionine-[S,R]-sulfoximine (BSO), hydroxocobalamin (vitamin B12a), and other reagents were purchased from Sigma Chemical (Poole, UK). Trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazol (SB-239063) was a kind gift from GlaxoSmithKline Laboratories (Stevenage, UK).
Cell culture. The transformed human bronchial epithelial cell line BEAS-2B was grown in K-SFM supplemented with 25 mg of BPE and 2.5 µg of EGF. Cells were cultured at 37°C in a humidified atmosphere containing 5% (vol/vol) CO2 in air. Cells were growth factor starved for 24 h before treatment with SIN-1 and/or MnTMPyP (1-20 µM), hydroxocobalamin (0.1-1 mM), PD-98059 (10-100 µM), and SB-239063 (0.1-10 µM). In some experimental conditions, BSO, an inhibitor of glutathione (GSH) synthesis, was supplemented to culture (1 mM) 24 h before exposure to the stipulated reagents.
Cell viability. Cell viability was quantified by CCK-8 assay according to the manufacturer's instructions. In brief, cells were seeded in 96-well plates and then incubated in K-SFM for 24 h with or without 1 mM BSO. Cells were treated with reagents from 8 to 48 h in complete media or K-SFM, and 10 µl of CCK-8 reagent were added to each well. Plates were incubated from 30 min to 2 h at 37°C, and the difference in absorbance between 450 and 600 nm was measured as an indicator of cell viability. Control cells were treated in the same way with diluents when necessary, and the value of different absorbance was defined as 100% survival.
Measurement of ONOO generation.
ONOO
generation from SIN-1 was measured essentially by
the method of Haddad et al. (14), whereby production of
ONOO
was assessed by oxidation of dihydrorhodamine 123 to
rhodamine, and absorbance was measured at 500 nm.
Western blotting. Cells treated with reagents in six-well plates were scraped into 1 ml of Hanks' balanced salt solution and centrifuged at 1,000 g for 5 min. The cell pellets were lysed with 10 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 1% (vol/vol) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin (lysis buffer) for 30 min in ice. The lysed cells were then centrifuged at 16,000 g for 15 min. The protein concentration of the resulting supernatant was determined by the Bio-Rad protein assay system according to the manufacturer's instructions.
Equal amounts of protein (20-80 µg) were resolved with 4-12% precast Bis-Tris gels. After transferring the proteins onto a nitrocellulose membrane, we blocked the membranes with 5% (wt/vol) nonfat milk in phosphate-buffered saline solution containing 0.1% (vol/vol) Tween 20 for 1 h at room temperature. The blot was then incubated with antibodies specific for either phosphorylated or total ERK, p38, and JNK (1/1,000 dilution). The membrane was then treated with appropriate secondary antibody conjugated with horseradish peroxidase and visualized by chemiluminescence with ECL. In some experiments, we probed blots for nitrotyrosine formation using an antinitrotyrosine antibody (1/x) dilution.In vitro MAPK assay.
The in vitro activity of ERK and p38 was measured with a p44/42 or p38
MAPK assay kit (New England Biolabs). Cellular proteins (40 µg) were
incubated with either immobilized phospho-p44/42 MAPK
(Thr202/Tyr204) monoclonal antibody or
immobilized phospho-p38 MAPK (Thr180/Tyr182)
monoclonal antibody overnight at 4°C to precipitate ERK or p38, respectively. After washing the pellet, we performed an in vitro kinase
reaction at 30°C for 30 min in kinase buffer [25 mM Tris pH 7.5, 5 mM -glycerolphosphate, 2 mM dithiothreitol (DTT), 0.1 mM
Na3VO4, and 10 mM MgCl2]
containing 200 µM ATP and either glutathione S-transferase
(GST)-Elk-1 (307-428) (substrate for ERK) or
GST-activating transcription factor (ATF)-2 (19-96)
(substrate for p38) fusion proteins. The reaction was terminated by the
addition of 3× SDS sample buffer [187.5 mM
Tris · HCl pH 6.8, 6% (wt/vol) SDS, 30% (vol/vol) glycerol, 150 mM DTT, and 0.3% (wt/vol) bromphenol blue]. The samples were then boiled for 3 min, and the proteins were resolved
with a 4-12% (wt/vol) Bis-Tris gel. Transfer of proteins onto
nitrocellulose, membrane blocking, and antibody incubations were as
described above with antibodies specific for phosphorylated Elk-1
(Ser383) or phosphorylated ATF-2 (Thr71)
(1/1,000).
Statistical analysis. Data are presented as the means ± SE for n determinations. Differences were analyzed by one-way ANOVA or Mann-Whitney's tests as appropriate. P < 0.05 was considered significant.
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RESULTS |
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Effects of SIN-1 on BEAS-2B cell viability.
The addition of 1 mM SIN-1 reduced the viability of BEAS-2B cells in a
time- and dose-dependent manner, as determined by CCK-8 assay.
Twenty-four-hour treatment of BEAS-2B cells with 1 mM SIN-1 led to a
significant reduction in cell viability to ~50% of control levels
and could be reduced further after 48 h to 30% (Fig.
1A). To determine whether
SIN-1-induced cell death involved oxidative mechanisms, we pretreated
cells with 1 mM BSO, an inhibitor of the GSH synthesis, for 24 h.
This treatment enhanced the SIN-1-mediated loss of cell viability such
that at 8 h post-1 mM SIN-1 exposure, only ~17% of control
cells remained viable (Fig. 1A). The pretreatment of BEAS-2B
cells with 1 mM BSO alone for 24 h was not cytotoxic (data not
shown). To further examine this effect of SIN-1, we exposed cells to
increasing concentrations of SIN-1 for 8 h in the absence or
presence of growth factors. Concentrations of SIN-1 of 0.25-1 mM
in K-SFM supplemented with growth factors (Fig. 1B) and
0.25-1 mM in K-SFM (Fig. 1B) elicited a significant
reduction in cell viability.
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Time course effects of SIN-1 on MAPK activation.
To examine whether SIN-1 could mediate MAPK activities in
BEAS-2B cells, we immunoblotted whole cell lysates using antibodies against phospho-ERK, phospho-p38, or phospho-JNK. SIN-1 treatment in
BSO-pretreated BEAS-2B cells activated all three MAPK subgroups; however, the kinetics of the responses were different. The level of
phosphorylated ERK began to increase 1 h following 1 mM SIN-1 treatment, reached a maximum at 8 h, then persisted at 24 h
(Fig. 3A). This result was
confirmed by in vitro ERK kinase assay using GST-Elk-1 as a substrate
of phospho-ERK and revealed by immunoblotting with phospho-Elk-1
antibody. Similarly, the phosphorylation of p38 also increased after
1-h treatment with 1 mM SIN-1, reached a maximum at 4 h, then
decreased to the basal level after 8 h (Fig. 3B). An in
vitro p38 kinase assay using GST-ATF-2 as a substrate of phospho-p38
and phospho-ATF-2 antibody to measure p38 activity demonstrated a
similar kinetic profile. In contrast, the phosphorylation of JNK was
delayed compared with ERK and p38, starting 2 h after SIN-1
treatment, before reaching a maximum at 4 h, then returning to the
basal level after 8 h (Fig. 3C). Phosphorylated ERK,
JNK, and p38 did not change in control cells during the 24-h time
course (data not shown). Similar experiments were performed on cells that had not been pretreated with BSO; under these conditions there was
no activation of the MAPK pathways (data not shown). This strongly
suggests that these observations are dependent on oxidant stress of the
epithelial cells.
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Dose-dependent effects of SIN-1 on MAPK activation.
Having determined the time course of MAPK activation with SIN-1, we
performed experiments using the appropriate time points to determine
whether this effect was concentration dependent. ERK phosphorylation
and activity increased dose dependently following 2 h of treatment
of BEAS-2B cells with SIN-1 (Fig.
4A). Likewise, phosphorylation
of p38, p38 kinase activity, and phosphorylation of JNK in the presence
of SIN-1 was also increased dose dependently (Fig. 4, B and
C). We determined the possibility that activation of MAPK
was not due to ONOO but to the alternative product of the
reaction, SIN-1C, by exposing cells to media that had contained SIN-1
for 24 h. At this time point, ONOO
is no longer
generated; however, this medium did not alter the levels of
phosphorylated ERK (data not shown).
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Effects of NO · and
O and not NO · and/or
O
and are not due to the effects of
NO · or O
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Effects of MAPK pathway inhibitors on SIN-1-induced MAPK
activation.
To determine whether SIN-1-induced ERK activity was a MEK-dependent
mechanism, we cultured BEAS-2B cells in the presence of PD-98059, an
inhibitor of MEK. Cells were treated with 50 µM PD-98059 30 min
before exposure with 1 mM SIN-1 for 2 h. This treatment inhibited
partially SIN-1-induced ERK activity as determined by phosphorylation
immunoblotting and in vitro kinase assay (Fig. 6A). Furthermore, a selective
inhibitor of p38, SB-239063, was also able to inhibit SIN-1-induced p38
activity (Fig. 6B).
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Effects of MAPK pathway inhibitors on SIN-1-induced cell death.
Because SIN-1 could activate the MAPK pathways, the effects of MAPK
inhibitors on the SIN-1-induced cell death were examined. PD-98059 and
SB-239063 failed to inhibit SIN-1 (1 mM)-induced cell death (Fig. 7,
A and B),
suggesting that the effects of SIN-1 on the MAPK pathways might be
unrelated to the effects on cell survival. To confirm whether this was
in fact the case, we repeated the experiment using 0.25 mM SIN-1 for
8 h. Under these experimental conditions, PD-98059 significantly
attenuated SIN-1-induced cell death (60% cell survival at the
concentration of 25 µM), whereas SB-239063 had no effect (Fig. 7,
C and D), suggesting that this effect of SIN-1 is
mediated via ERK and not p38. However, when both these inhibitors were
used together, SB-239063 potentiated the effects of PD-98059 on
SIN-1-induced cell death with cell viability (Fig.
8A). These data suggested an
interaction between the ERK and p38 MAPK pathways. To examine this
possibility further, we treated cells with PD-98059 and determined p38
MAPK phosphorylation after 4-h SIN-1 exposure. However, PD-98059 failed
to inhibit SIN-1-induced p38 phosphorylation (Fig. 8B). In
contrast, when cells were treated with the p38 inhibitor SB-239063 in
the presence of SIN-1, ERK activation was reduced, suggesting cross
talk between these pathways (Fig. 8C).
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DISCUSSION |
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Several lines of evidence suggest a role for ONOO in
the pathophysiology of chronic inflammatory diseases, including asthma and COPD (35, 38). The present study examined the effects of SIN-1, a ONOO
generator, on proliferation of human
bronchial epithelial cells. This study reports that SIN-1 exposure led
to a dramatic cell death. Furthermore, it appeared that this effect was
associated with the activation of the three major subgroups of MAPK
pathways: ERK, JNK, and p38 MAPK. This was further supported by data
that showed that inhibition of ERK and p38 MAPK pathways could reduce the cell death induced by SIN-1.
ONOO-inducible cell death has been demonstrated in
neuroblastoma cells where SIN-1 treatment led to cytotoxicity after
24-h exposure by apoptosis measured with flow cytometric
analysis and the activation of caspase-3 protease (31,
32). Similar effects of ONOO
have also been
observed in HL-60 and neuronal cells (28, 42). In the
lung, cell death induced by ONOO
has been demonstrated in
rat type II epithelial cells, bovine pulmonary artery endothelial
cells, and rat pulmonary myofibroblasts, but not in human bronchial
epithelial cells (12, 33, 47). This study showed that
SIN-1-induced cell death occurred through an oxidative pathway, since
inhibition of GSH synthesis accelerated cell death. Recently, an
increased vulnerability of neuroblastoma SH-SY5Y cells with SIN-1
exposure after depletion in GSH by BSO pretreatment was reported
(31). This may be important since GSH depletion has been
linked to the pathophysiology of many airway diseases, including
idiopathic pulmonary fibrosis, acute respiratory distress syndrome,
bronchopulmonary dysplasia, and cystic fibrosis (3, 4, 34,
39). SIN-1 is thought to release simultaneously NO · and O
. MnTMPyP, a scavenger of O
, but not
O
The mechanism of these ONOO-mediated events is unclear,
since ONOO
production via SIN-1 is back to baseline
levels at 8 h. This suggests that ONOO
is causing a
cascade of events leading to cell death at 8 h. ONOO
may lead to nitration of tyrosine residues of proteins and this may
alter phosphotyrosine-dependent signaling. However, not only can
ONOO
prevent phosphorylation of tyrosine residues by
nitration, but also such covalent modification of tyrosine residues may
induce phosphorylation. Such a mechanism has been reported for src
tyrosine kinases, Akt kinase, and EGF receptor in some cell types
(24, 29, 47). Furthermore, ONOO
can
activate MAPK pathways in a variety of cells such as neuronal cells,
polymorphonuclear leukocytes, liver epithelial cells, or fibroblasts
(1, 21, 26, 40). The data presented in this study
demonstrate for the first time that SIN-1 can activate the three major
subgroups of MAPKs in human bronchial epithelial cells in both a time-
and concentration-dependent manner. Moreover, the inhibition of
activation of MAPKs induced by SIN-1 with either MnTMPyP or
hydroxocobalamin treatments suggests the involvement of
ONOO
in this response. It is unlikely that this is a
direct effect of ONOO
on MAPK, since the effects on
activity are not evident for hours following stimulation. This suggests
that ONOO
may be mediating upstream effectors in the
activation pathway; nevertheless, it is ONOO
and not
NO · or O
-mediated cell death have been
suggested, including disruption of mitochondrial function
(46), which can lead to release of cytochrome c
and activation of the caspase pathway (37). Such mitochondrial disruption can also alter ATP levels in the cell, contributing to cell death (14). Recently, it has been
suggested that lipid peroxidation via the action of ONOO
may also be contributing to cell death in pulmonary epithelial cells
(17).
Because SIN-1 could activate the MAPK pathways, inhibitors of both ERK
or p38 MAPK pathways were investigated to determine whether MAPK were
responsible for this observation. Neither PD-98059, an inhibitor of MEK
(7), nor SB-239063, a selective inhibitor of p38
(43), had any effect on cell survival after treatment with
1 mM SIN-1. These data suggest that there was no interaction between
these two effects of SIN-1 in epithelial cells. The possibility that 1 mM SIN-1 is a supramaximal concentration to test these inhibitors was
addressed by use of a lower concentration of SIN-1. Treatment of
BEAS-2B cells with 0.25 mM SIN-1 led to a cytotoxicity of 60% compared
with control cells after 8-h exposure. The MEK inhibitor PD-98059
partially enhanced cell survival, whereas SB-239063, a p38 MAPK
inhibitor, was not able to protect cells against SIN-1 exposure. These
results suggest that the ERK pathway is as least in part involved in
SIN-1-induced cell death. In most systems studied, the ERK pathway is
activated in response to mitogenic factors and is generally poorly
stimulated by stress stimuli. However, the protective effect of
PD-98059 against ONOO treatment has been reported in
neuroblastoma cells and pulmonary myofibroblasts, indicating that the
ERK pathway is able to transduce cell death signals of
ONOO
exposure (32, 36, 47). This has been
confirmed in this study of human bronchial epithelial cells. Moreover,
treatment of BEAS-2B cells with both PD-98059 and SB-239063 inhibited
SIN-1-induced cell death, leading to >80% of cell survival. This is
of interest because it provides evidence of an interaction between the
ERK and p38 MAPK pathways in the response to SIN-1 in human bronchial epithelial cells. In addition, we show a decrease of phosphorylated ERK
induced by SIN-1 with the inhibition of p38 MAPK, suggesting that ERK
activation by SIN-1 was partially mediated by p38 MAPK. Recently,
Houliston et al. (18) reported cross talk between ERK and
p38 MAPK pathways, where SB-203580 treatment could enhance IL-1
-induced ERK but reduced thrombin-stimulated ERK pathway in
human umbilical vein endothelial cells, suggesting a differential signal upstream ERK pathway. A similar mechanism may be responsible for
this observed result.
In conclusion, this study demonstrates that ONOO can
affect MAPK signaling pathways in human bronchial epithelial cells and that their inhibition could protect cells against the oxidative stress
induced by ONOO
. Such stresses could occur in the
inflamed airway due to the induction of inducible nitric oxide
synthase, which could produce NO, in association with an increase in
inflammatory cells, such as neutrophils and macrophages, which could
produce superoxide. This is the case in diseases such as asthma and
COPD, where exhaled levels of NO are increased and there is an increase
in inflammatory cells in the airway (15, 22, 23, 30). This
effect may be relevant in inflammatory airway diseases such as asthma,
where the airway epithelium may be exposed to ONOO
, which
may then in turn lead to the epithelial damage and loss, which is
characteristic of asthma, thus prolonging the cycle of inflammation
observed in such diseases.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the National Asthma Campaign, UK, and Pharmacia.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. E. Donnelly, Dept. of Thoracic Medicine, National Heart and Lung Institute, Faculty of Medicine, ICSTM, Dovehouse St., London SW3 6LY, UK (E-mail: l.donnelly{at}imperial.ac.uk).
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.
First published February 21, 2003;10.1152/ajplung.00178.2002
Received 6 June 2002; accepted in final form 13 February 2003.
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