Department of Pathology, University of Vermont, Burlington, Vermont 05405; and Department of Anesthesiology, University of Alabama, Birmingham, Alabama 35294
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ABSTRACT |
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Reactive oxygen
(ROS) or nitrogen (RNS) species can affect epithelial cells to cause
acute damage and an array of pulmonary diseases. The goal of this study
was to determine patterns of early response gene expression and
functional end points of exposure to nitric oxide (NO ·),
H2O2,
or peroxynitrite (ONOO)
in a line of rat lung epithelial (RLE) cells. Our focus was on
c-fos and
c-jun protooncogenes, as these genes
play an important role in proliferation or apoptosis, possible end
points of exposure to reactive metabolites in lung. Our data
demonstrate that NO · generated by spermine
1,3-propanediamine
N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl} or
S-nitroso-N-acetylpenicillamine
as well as
H2O2
cause increased c-fos and
c-jun mRNA levels, nuclear proteins,
and complexes binding the activator protein-1 recognition sequence in
RLE cells. These agents also lead to apoptosis and increased membrane
permeability. In contrast, exogenously administered
ONOO
or
3-morpholinosydnonimine do not induce protooncogenes or apoptosis in
RLE cells despite nitration of tyrosines. We conclude that ROS and RNS
can elicit distinct molecular and phenotypic responses in a target cell
of pulmonary disease.
nitric oxide; peroxynitrite; hydrogen peroxide; lung epithelium; reactive oxygen species; reactive nitrogen species
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INTRODUCTION |
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EPITHELIAL CELLS of the lung are exposed to a variety of free radical species both after inhalation of oxidant gases and other pollutants and as a consequence of inflammation (6, 11, 12, 26). The chemistry of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is complex and involves the formation of a number of intermediate species that may interact differently with biological targets (2). Formation of ROS and RNS and their reactions with target cells of disease are thought to be linked to the initiation of a number of pulmonary disorders, including pulmonary fibrosis, acute respiratory distress syndrome, bronchiolitis, asthma, and lung cancer (6, 11, 12, 15).
The identity of reactive species causing alterations in gene expression
and biological responses in target cells of disease in the lung and
other organs still remains uncertain. Likely candidates include
H2O2,
nitric oxide (NO ·), and peroxynitrite
(ONOO), which is formed
from the rapid reaction of superoxide
(
) with NO ·.
ONOO
is an extremely potent
oxidant that can cause lipid peroxidation, DNA damage, and alterations
of protein function in vitro (2, 15). However, the effects of ROS and
RNS on early response gene expression and phenotypic consequences in
pulmonary target cells of disease and other cell types have not been
investigated. One goal of these studies was to determine whether
c-fos and
c-jun protooncogenes are activated
selectively by these reactive species in rat lung type II alveolar
epithelial (RLE) cells. Although we and others have shown the rapid
induction of c-fos and
c-jun in other cell types after
exposure to
H2O2
or NO · (14, 17, 22, 23), nothing is known about induction of
early response protooncogenes by
ONOO
or the phenotypic
consequences of gene expression by
ONOO
. We therefore used a
number of chemical generators of NO ·,
ONOO
, or
H2O2
to determine whether these oxidants could activate these molecular
events and apoptosis in an RLE cell line. We also wanted to determine
whether responses were unique to the type of reactive species
encountered or general phenomena observed in oxidative stress.
The c-fos and
c-jun genes encode proteins that can
dimerize to form homodimeric (Jun/Jun) and heterodimeric (Fos-Jun)
complexes of the activator protein (AP)-1 family, accessory
transcription factors that interact with DNA regulatory sequences known
as 12-O-tetradecanoylphorbol-13-acetate response elements or
AP-1 sites (1). In this study, we comparatively measured
c-fos and
c-jun mRNA levels, Fos and Jun nuclear
proteins, and increases in AP-1 DNA binding activity in RLE cells
exposed to NO ·,
H2O2,
or ONOO. In addition,
because early response gene transactivation has been linked to the
development of apoptosis, a unique type of programmed cell injury, in
other cell types (24), we measured apoptosis and membrane damage as
biological consequences of exposure to RNS or ROS in epithelial cells
of the lung.
Our results show that exogenously administered NO · or
H2O2,
but not ONOO, causes
increases in c-fos and
c-jun gene and protein expression, AP-1 DNA binding activity, apoptosis, and membrane permeability. Our
results also demonstrate that, despite protein tyrosine nitration, no
effects were observed with
ONOO
.
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EXPERIMENTAL PROCEDURES |
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Cell culture and exposure to test agents. A rat lung type II epithelial cell line that retains its normal differentiated features and near-diploid karyotype (RLE-6TN) was propagated as described previously (8). Cells were grown to confluency, and medium was switched to 1% serum containing medium for 24 h before addition of test agents.
Synthetic ONOO was
manufactured by the reaction of
H2O2
with NaNO2, and excess
H2O2
was removed by treatment with MnO2
according to procedures described elsewhere. 3-Morpholinosydnonimine
(SIN-1; Molecular Probes, Eugene, OR) was used as a continuous
generating system for
ONOO
as this compound
undergoes a base-catalyzed reaction leading to the simultaneous release
of
and NO ·,
which rapidly react to form
ONOO
(2). Spermine
1-3-propanediamine
N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl} (NONOate) (Cayman Chemical, Ann Arbor, MI) was used as an
NO · donor. Alternatively,
S-nitroso-N-acetylpenicillamine
(SNAP; Molecular Probes) in the presence of L-cysteine (100 µM; GIBCO) and SIN-1 in the presence of superoxide dismutase (SOD,
300 U/ml; Calbiochem, San Diego, CA) were used to confirm results
obtained with spermine NONOate. All NO · generators were
dissolved in Hanks' balanced salt solution (HBSS) and were added to
cell cultures immediately. H2O2
was purchased from Sigma (St. Louis, MO). Cells were exposed to these
agents in 1% serum containing medium employing a range of
concentrations that varied from 50 µM to 1 mM. As a reagent control
for ONOO
, cells were
exposed to inactivated ONOO
after a 24-h incubation at room temperature.
ONOO
was quantitated at 302 nm using an extinction coefficient of 1,670 M
1 · cm
1.
Measurement of NO · in RLE cell medium after exposure to NO ·-generating systems. Evolution of NO · in the medium by spermine NONOate, SNAP plus L-cysteine (100 µM), or SIN-1 in presence of SOD (300 U/ml) was measured in HBSS with an ISO-NO electrochemical probe (WPI) connected to a chart recorder as described elsewhere (16). The ISO-NO meter was calibrated by the addition of a NO ·-saturated (1.8 mM) solution.
Isolation of RNA and Northern blot
analyses. At selected time periods after exposure to
ONOO,
NO ·-generating systems, or
H2O2,
cells were harvested for extraction of total RNA. RNA was
electrophoresed on 3-(N-morpholino)propanesulfonic acid
formaldehyde gels and was transferred onto nitrocellulose. Blots were
hybridized with random primed
[32P]cDNA probes (28).
cDNAs encoding c-fos and
c-jun were obtained from R. Gaynor at
the University of California at Los Angeles. Blots were washed in
standard sodium citrate (28) and were exposed to film (NEF 496; NEN,
Boston, MA). Quantitation was performed using a PhosphoImage Analyzer
(Bio-Rad, Hercules, CA).
Western blotting of c-Fos and c-Jun proteins. Nuclear extracts prepared from RLE cells, as described previously, were diluted in 2× Laemmli sample buffer (28, 29). Samples (5 µg/lane) were electrophoresed on a 15% polyacrylamide gel and were electroblotted onto nitrocellulose (Ellard Instrumentation, Seattle, WA) according to standard procedures (28). Blots were incubated overnight at 4°C in tris(hydroxymethyl)aminomethane (Tris)-buffered salt (TBS) solution containing 5% nonfat milk. Membranes were then washed with TBS containing 0.05% Tween 20 for 30 min followed by incubation with c-Fos or c-Jun antibodies for 1 h (0.5 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were washed and then were incubated with peroxidase-conjugated secondary antibody (0.3 µg/ml; Jackson Immunoresearch Laboratories, West Grove, PA). Proteins were visualized by enhanced chemiluminescence (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Electrophoretic mobility shift
analyses. To determine binding of AP-1 transcription
factor complexes to the AP-1 consensus DNA sequence (Promega, Madison,
WI), nuclear extracts from RLE cells treated with
ONOO,
NO ·-generating systems, or
H2O2
were prepared according to procedures by Staal et al. (29). Four
micrograms of nuclear protein were applied per lane and were
electrophoresed on 4% polyacrylamide gels in 0.25×
Tris-borate-EDTA buffer at 100 V for 2 h. Gels were dried and exposed
to film at room temperature. Afterward, the shifted bands containing
the AP-1 complex were quantitated by phosphoimage analysis.
Measurement of cell cycle distribution and
apoptosis. Flow cytometry was performed on cell
cultures exposed to ONOO,
NO ·-generating systems, or
H2O2
for 24, 48, or 72 h. Total cells (attached plus floating) were
harvested by brief trypsinization and were resuspended in 3.75 mM
sodium citrate, pH 7.0, 0.1% Triton X-100, 32 µg/ml ribonuclease A,
and 50 µg/ml propidium iodide (Sigma Chemical) by methods described
previously (3). Ten thousand gated events per group per experiment in
duplicate were evaluated to determine the percentage of cells in
G0/G1,
S, and G2/M phases of the cell
cycle and those exhibiting a hypodiploid DNA content characteristic of
apoptosis (7).
To also visualize the occurrence of DNA laddering, which is
characteristic of apoptosis, genomic DNA was extracted from cells exposed to ONOO,
NO ·-generating systems, or
H2O2
for 24 h (5). Thirty micrograms of DNA were loaded on a 1.6% agarose
gel containing ethidium bromide and were electrophoresed in
Tris-borate-EDTA buffer at 20 V overnight. Gels were then photographed
under ultraviolet light.
Assessment of membrane permeability. To determine whether test agents caused membrane damage, the fluorescent dye Sytox (Molecular Probes), which selectively enters cells with permeable membranes, was used. After 24 h of exposure to agents, cells were trypsinized from dishes and incubated in 10 nM Sytox in HBSS (GIBCO), and ten thousand cells per group were analyzed by flow cytometry (Coulter EPICS Elite, Miami, FL). Numbers of fluorescent cells correlated with numbers staining positively using the trypan blue exclusion technique to assess viability (data not shown).
Immunofluorescence of nitrotyrosine
residues. RLE cells were grown on glass coverslips and
were treated with test agents in 1% serum-containing medium as
described before. After 4 h of exposure, cells were washed two times
with phosphate-buffered saline (PBS) and were fixed in 100% methanol
(20°C) for 5 min. After three washes with PBS (5 min each),
cells were permeabilized for 20 min in PBS containing 0.1% Triton
X-100. Sections were washed in PBS containing 1% bovine serum albumin
and were incubated for 1 h with a rabbit polyclonal anti-nitrotyrosine
antibody (2 µg/ml PBS; Upstate Biotechnology, Lake Placid,
NY), washed with PBS, and incubated with a
lissamine-rhodamine-conjugated goat anti-rabbit secondary antibody (20 µg/ml; Jackson Immunoresearch Laboratories) for 1 h. Coverslips were
mounted onto slides using Vectashield mounting medium (Vector
Laboratories, Burlingame, CA) for analysis by confocal microscopy
(Bio-Rad).
Statistical analysis. Results were evaluated by one-way analysis of variance using the Student-Newman-Keuls procedure for adjustment of multiple comparisons. Trend analysis was performed to assess the dosage dependence of responses.
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RESULTS |
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Production of NO · in medium. The maximal concentrations of NO · measured over a period of 2 h were 15 µM for 1 mM spermine NONOate and 30 µM for 1 mM SNAP in the presence of 100 µM L-cysteine. One millimolar SIN-1 in the presence of 300 U/ml SOD yielded ~0.8-1 µM NO · similar to values reported previously (16), whereas no NO · could be measured in the absence of SOD. It is of importance to note that the rate of NO · formation in medium differs between these compounds. The maximal NO · concentrations were found after 10 min of exposure to SNAP plus L-cysteine, which decreased by 50% after 20 min and disappeared after 2 h. However, spermine NONOate and SIN-1 plus SOD released constant levels of NO · over a time period encompassing ~2 h, which disappeared after 4 h of exposure. The difference in kinetics in NO · generation over time may explain the differences in magnitude of responses observed in RLE cells exposed to NO · generators (see below).
H2O2 and
NO ·, but not ONOO, cause
increased c-fos and
c-jun mRNA levels, protein, and AP-1 DNA
binding activity.
Because the fos and
jun gene families appear to regulate a
number of cellular events, including proliferation and apoptosis, we
first determined whether various NO ·-generating systems,
ONOO
, or
H2O2
could induce c-fos and
c-jun and their protein products. Figure
1A
demonstrates increases in steady-state mRNA levels of c-fos and
c-jun after 2 h of exposure of RLE
cells to
H2O2
or spermine NONOate. With these agents, increases in mRNA levels appeared after 1 h of exposure, were maximal at 2 h, and decreased after 4 h of exposure (data not shown). Exposure to
ONOO
(150 µM-1 mM) did
not alter mRNA expression of c-fos or
c-jun at any time point. To avoid the
possibility that components in newborn bovine serum may have scavenged
ONOO
, we also examined
c-fos and
c-jun expression in cells after
exposure to ONOO
in
serum-free medium and obtained similar negative results (data not
shown). Figure 1B shows
c-jun mRNA levels after 2-h exposures to a range of NO · generators. Like spermine NONOate, SNAP,
in the presence of L-cysteine, caused statistically
significant increases in expression of
c-jun. The
OONO
generator SIN-1 did
not affect c-jun mRNA, similar to
findings with synthesized
ONOO
. However, SIN-1, in
the presence of SOD, a protocol generating NO · and
H2O2,
augmented c-jun mRNA levels
(P < 0.05).
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Differential induction of apoptosis and membrane permeability by
H2O2,
NO ·, and ONOO.
Because early response protooncogenes may be important in the
activation of the apoptotic pathway (24), we next determined whether
exposure to NO ·-generating systems,
ONOO
, or
H2O2
induced apoptosis or increases in membrane permeability. We first used
flow cytometry to determine the fractions of RLE cells in different
phases of the cell cycle. After a 24-h exposure to spermine NONOate,
SNAP, SIN-1 in the presence of SOD, or
H2O2, a significant (P < 0.05) percentage
of cells exhibiting a hypodiploid DNA content indicative of apoptosis
was apparent (Fig.
5A).
Numbers of apoptotic cells increased over a 72-h period (data not
shown). RLE cells exposed to
ONOO
did not display
alterations in normal cell cycle distributions or increases in
apoptosis at any time point. We next exposed cells to various
concentrations of agents to confirm these observations. As demonstrated
in Fig. 5B, exposure to spermine
NONOate or
H2O2 caused dosage-dependent increases in apoptosis, whereas synthetic ONOO
or the
ONOO
generator SIN-1 did
not. As shown in Fig. 6, DNA ladders
appeared in RLE cells after exposure to spermine NONOate, SNAP, or
H2O2. No evidence of DNA degradation was present in RLE cells exposed to
SIN-1. Similar to results by flow cytometry, SIN-1, in the presence of
SOD, caused the typical laddering pattern that is characteristic of
apoptosis. Although synthesized
ONOO
did not cause discrete
laddering of DNA, some smearing was evident, which may be indicative of
nonspecific cleavage of base pairs. In support of data obtained by flow
cytometry and DNA laddering, assessment of nuclear morphology using
4',6-diamidino-2-phenylindone showed characteristic apoptotic
bodies in RLE cells after exposure to
H2O2
or NO ·-generating systems, but not
ONOO
(data not shown).
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DISCUSSION |
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Studies here indicate that both H2O2 and NO · serve as important intracellular signaling molecules to activate protooncogene expression and transcription factors. Our focus here was on c-fos and c-jun, since protein products of these protooncogenes are thought to play a role in cell differentiation, transformation, proliferation, and apoptosis (1, 24, 25). These events may be critical to the development of pulmonary disease associated with exposure to reactive metabolites. Although others have demonstrated activation of mitogen-activated protein kinases by H2O2 (13, 25) and NO · (20), suggesting that early response genes may be downstream targets, we demonstrate here that H2O2 and a variety of NO ·-generating systems activate c-fos and c-jun protooncogenes, as evidenced by increased mRNA expression, nuclear proteins, and AP-1 DNA binding activity. Furthermore, H2O2 or NO · also lead to the development of apoptosis, as defined by flow cytometry, DNA laddering, and morphological criteria for apoptosis.
Increased mRNA levels of fos and
jun family members and activation of
AP-1-dependent gene expression occurs in other cell types after
exposure to NO · or
H2O2
(14, 17, 22, 23). In addition, NO ·-generating systems (4,
18) and
H2O2
(3) cause apoptosis in a variety of cell types, including pancreatic -cells and cortical and mesothelial cells. Although our studies do
not provide a direct link between early response gene activation and
apoptosis, the time frame of events and our negative results using
ONOO
suggest a possible
association. Present studies that involve overexpression of c-Fos or
c-Jun in RLE cells may verify the association of these protooncogenes
with apoptosis.
NO · can react with a number of substrates that may dramatically alter its reactivity and toxic potential. For instance, reaction with heme-containing proteins may alter their function (2), whereas reaction with thiol moieties in airways may increase the half-life of NO · by providing a continuous source of NO · release from nitrosothiols (12). At high concentrations, NO · reacts with O2 to form NO2 · (2). At the peak concentrations of NO · generated here, small levels of NO2 · may have been formed and may have accounted for some of the observed effects. Because NO2 · is also a nitrating agent, the lack of nitrotyrosine immunofluorescence after exposure to NO · generators (not shown) indicates that NO2 · levels were minimal.
The reaction of NO · with
that generates the potent
oxidant, ONOO
, is extremely
rapid, and, in situations where elevated NO · concentrations exist, NO · outcompetes SOD for reaction with
(2). In inflammatory
conditions or ischemia-reperfusion,
and NO · can be
generated simultaneously from different cell types (26), thus providing
substrates for ONOO
generation. ONOO
, in its
physiological, protonated form, also is extremely reactive and
decomposes into a number of reactive metabolites that may involve
NO2 · and OH · radicals (2), which may contribute to oxidative DNA modification (31),
alterations in protein function (30), or lipid peroxidation (27). Thus
the overall reaction chemistry of RNS and ROS and the critical reactive
species that ultimately damage lung cells are extremely complex.
Exogenous administration of
ONOO at a range of
concentrations (150 µM-1 mM) causing nitration of tyrosines (Fig. 8)
did not lead to alterations in early response gene expression or
apoptosis in RLE cells when assessed by flow cytometry, DNA laddering,
and nuclear staining techniques. The lack of increased membrane
permeability by ONOO
confirmed these findings and further substantiated its inability to
induce injury in this cell type. The use of SIN-1 as another exogenous
generating system of ONOO
supported these findings. However, incubation of SIN-1 in the presence
of SOD to yield NO · and
H2O2
caused protooncogene induction, apoptosis, and membrane damage,
suggesting that NO · and
H2O2, but not ONOO
, induce
signaling events that lead to these phenotypic end points. These
findings, however, contrast with other studies showing apoptosis in
PC-12 cells (9), cerebrocortical cultures (4), and HL-60 cells (21)
exposed to ONOO
. However,
in support of our findings, a recent study demonstrates that
ONOO
generated by SIN-1
does not cause cytotoxicity, as measured by lactic dehydrogenase
release in a human ovarian cancer cell line (10). In contrast, SIN-1
plus SOD resulted in enhanced toxicity, suggesting a role for
NO · and
H2O2
in the cytotoxic effect (10). This work and the lack of responsiveness
of RLE cells to ONOO
is
surprising given the reactivity of this species (2). However, a number
of possible explanations exist. First, signaling cascades that lead to
activation of c-fos and
c-jun and consequent apoptosis may be
inactivated by ONOO
. For
example, it was demonstrated recently that nitration of tyrosine
residues by ONOO
may block
phosphorylation (19), thereby impairing tyrosine kinase signaling
cascades that may be required for protooncogene activation or
apoptosis. Alternatively, externally administered ONOO
may react with cell
surface components, as is indicated by patterns of tyrosine nitration,
whereas NO · or
H2O2
can traverse the cell membrane to elicit protooncogene expression.
However, sensitive transient transfection assays in RLE cells to
measure activation of gene transcription by nuclear factor-
B
(NF-
B) demonstrate that
ONOO
activates
NF-
B-dependent gene expression (unpublished observations).
In conclusion, our findings demonstrate that NO · or
H2O2
causes increased early response gene expression resulting in apoptosis in lung epithelial cells. In contrast,
ONOO did not cause these
responses. However, the lack of gene expression and apoptosis observed
here with ONOO
occurred in
the presence of nitrotyrosine residues, suggesting that their formation
per se cannot be equated with functional ramifications examined here.
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ACKNOWLEDGEMENTS |
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We thank Colette Charland (Flow Cytometry Facility, University of Vermont) for flow cytometric analysis, Dr. Douglas J. Taatjes (Director, Cell Imaging Facility, University of Vermont), Dr. Jean-Claude Pache (Hopital Cantonal Universitaire de Geneve, Geneva, Switzerland), Dr. Timothy Quinlan (University of Virginia) for assistance with confocal microscopy, Dr. Pamela Vacek (Dept. of Biometry and Biostatistics, University of Vermont) for statistical analyses, Eric Walsh and Judith Kessler for providing illustrations, Drs. Immad Y. Haddad and Sha Ju (Dept. of Anesthesiology, University of Alabama) for help with the NO · measurements, and Dr. B. Kalyanaraman (Medical College of Wisconsin) for helpful suggestions.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-39469, ES-06499, ES-07038 (to B. T. Mossman), HL-31197, and HL-51173 and by a grant form the Office for Naval Research (to S. Matalon).
Y. M. W. Janssen is a fellow of the Parker B. Francis Foundation for Pulmonary Research.
Address for reprint requests: Y. M. W. Janssen, Dept. of Pathology, University of Vermont, Medical Alumni Bldg., Rm. A-143, Burlington, VT 05405.
Received 22 April 1997; accepted in final form 1 July 1997.
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