(Received for publication, May 20, 1997)
From the The CardioPulmonary Research Institute, Departments of Pediatrics and Pulmonary and Critical Care Medicine, Winthrop-University Hospital, State University of New York at Stony Brook School of Medicine, Mineola, New York 11501
Oxidative insults that are lethal to epithelial
cells kill either via apoptosis or necrosis. Nuclear factor-B
(NF-
B) is a redox-sensitive transcription factor that is activated
by oxidative insult, and NF-
B activation can protect cells from
apoptosis. To test if NF-
B can protect from necrotic cell death
caused by high levels of molecular O2 (hyperoxia), we
exposed human alveolar epithelial (A549) cells to hyperoxia. NF-
B
was shown to be activated and was translocated to the nucleus within
minutes. Nuclear translocation persisted over the course of several
days, and the levels of NF-
B protein and mRNA increased as well.
In hyperoxia, NF-
B regulation was independent of mitogen-activated
protein kinase (MAPK). In sharp contrast, there was neither nuclear
translocation of NF-
B nor any increase in expression after exposure
to H2O2 at a concentration where this oxidant
induces both MAPK and widespread apoptosis. Despite the activation and
increased expression of NF-
B in hyperoxia, this oxidant remained
lethal to the cells. These observations confirm the notion that
apoptosis occurs in the absence of NF-
B activation but indicate that
protection from cell death by NF-
B is probably limited to
apoptosis.
Oxidative stress resulting from the toxic effects of reactive oxygen intermediates (ROI)1 plays an important role in the pathogenesis of many disease states including carcinogenesis, atherosclerosis, and inflammatory disorders (1, 2). Oxidative injury can also lead to cell death, and ROI can have a role in apoptotic cell death induced by nonoxidative insults (3-8). Direct oxidative injury often occurs as a consequence of ventilatory O2 therapy, which is used in the treatment of critically ill patients who cannot breath efficiently. This treatment typically requires supraphysiologic concentrations of O2 (hyperoxia), which results in an elevated level of ROI in many cell types of lung, the organ that receives the highest level of O2 exposure (9, 10). The lung is estimated to be composed of as many as 60 cell types, which complicates the study of pathways to cell death by hyperoxia. On the other hand, cultured lung epithelial cells provide simpler models for understanding certain aspects of pulmonary biology. We have recently reported that A549 cells (derived from human type II alveolar epithelial cells) and HeLa cells succumb to hyperoxia not via apoptosis but by necrosis. In contrast, lethal doses of the oxidants H2O2 or superoxide kill these epithelial cells via apoptosis (11). These observations indicate that the pathways to cell death may differ, depending on the oxidant and dose used.
Very recent reports from several laboratories show that apoptotic
cell death can be prevented by the expression of nuclear factor-B
(NF-
B) (12-15), a multisubunit transcription factor that rapidly
activates the expression of genes involved in inflammation, infection,
and stress (16). Taken together, these recent reports suggest that the
induction of NF-
B may be part of a survival mechanism used to escape
cell death (17). In this report, we examine the expression of NF-
B
in cells exposed to lethal concentrations of hyperoxia or
H2O2 and show that, despite the induction of
NF-
B by molecular O2, the cells do not escape death.
Human lung adenocarcinoma A549 cells (ATCC CCL 185) were cultured and maintained in 95% room air, 5% CO2 as described previously (11). Subconfluent cultures were used in all experiments. For hyperoxia treatment, cells were maintained in sealed humidified chambers flushed with 95% O2, 5% CO2. Media and gases were refreshed daily. Control cells were cultured in 95% room air, 5% CO2. For the H2O2 experiment, cells were cultured in 95% room air, 5% CO2, 5 mM H2O2. These cells are known to be relatively resistant to H2O2, and millimolar concentrations of H2O2 are necessary to induce apoptosis in the culture medium used (11).
ImmunofluorescenceAll procedures were carried out at room
temperature. Cells grown on coverslips were washed once with 1 × phosphate-buffered saline (PBS, Life Technologies, Inc., Gaithersburg,
MD) and fixed for 10 min in 10% formalin buffered in 1 × PBS.
Coverslips were then rinsed with 3 changes of PBS and incubated with
1% BSA solution (Panvera Corp., Madison, WI) for 10 min. Cells were
incubated with a 2.5 µg/ml (in 1% w/v BSA) anti NF-B p65 antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 30 min and washed with 3 changes of 1 × TBS (0.1 M Tris-Cl, pH 7.4, 0.15 M NaCl). Secondary antibody, sheep anti-rabbit
IgG-rhodamine (Boehringer Mannheim, Indianapolis, IN) was diluted in
1% BSA to a concentration of 27 µg/ml and incubated with coverslips
for 30 min. The coverslips were washed in excess water and mounted to
microscope slides, and the results were examined by immunofluorescence
microscopy using the UV-2A filter (Nikon Inc., Melville, NY) for
rhodamine.
Cell lysates were prepared according to the
procedures recommended by New England BioLabs (Beverly, MA). Protein
from each sample was loaded onto a 10 or 12% SDS-polyacrylamide gel
(Bio-Rad, Hercules, CA). After electrophoresis, proteins were
transferred to Immobilon-P membranes (Millipore, Bedford, MA). For MAPK
detection, blots were incubated with an antibody that recognizes only
the activated (phosphorylated) MAPK protein (New England BioLabs). Antibody was detected by the enhanced chemiluminescent (ECL) reagent, CDP-Star (Boehringer Mannheim), and exposing the filter to x-ray film.
For NF-B detection, the filter was incubated with rabbit anti-NF-
B p65 antibody as recommended by the supplier (Santa Cruz
Biotechnology).
Total RNA was purified from cells by
using RNA STAT-60 (TEL-TEST "B", Inc., Friendswood, TX) according
to the supplier suggestions. RNA from each sample was loaded onto a 1%
agarose gel containing 2.2 M formaldehyde. The gel was
electrophoresed in 1 × MOPS (Sigma) buffer and transferred to a
0.2 µm nylon membrane (Schleicher & Schuell, Keene, NH). The blots
were exposed to UV using a DNA Transfer Lamp (Fotodyne, New Berlin, WI)
and prehybridized at 50 °C for at least 4 h in solution
containing 50% formamide, 5 × SSC, 1% blocking solution
(Boehringer Mannheim), 0.1% SDS, 0.1% sarcosine, and 100 µg/ml
yeast tRNA. The filter was then hybridized with digoxigenin-labeled
dUTP (Boehringer Mannheim) probes overnight at 50 °C and washed as
recommended. The hybridized signal was detected by using
anti-digoxigenin antibody and developed in ECL solution (Boehringer
Mannheim) according to the supplier recommendations. The probes for
NF-B (full-length human p65 cDNA) and glyceraldehyde-3-phosphate dehydrogenase mRNA (full-length human cDNA) detection were
labeled with random primer labeling kit (Boehringer Mannheim).
Because the lung is the primary target of O2 toxicity, we utilized lung epithelial cells as a model to study signal transduction by injurious levels of hyperoxia and oxidants. In particular, A549 cells, which are derived from an adenocarcinoma of alveolar type II cells, have been extensively studied with respect to their responses to oxidants and other airborne insults (6, 18). In earlier studies, we have shown that cultured A549 cells exposed continuously to 95% O2 suffer cell death via necrosis and not by apoptosis. In contrast, lethal concentrations of H2O2 or superoxide cause apoptosis (11).
Because recent reports suggest that NF-B can prevent cell death, we
investigated the role of NF-
B in these two distinct modes of cell
death. A549 cells exposed to 95% O2 showed evidence of
cell swelling by 24 h, and the culture gradually died off over the
course of one week (11). It is known that following release from the
inhibitory binding protein I
B, NF-
B translocates from cytosol to
the nucleus, where it regulates transcription (19). We therefore
studied NF-
B activation during hyperoxia by examining its nuclear
translocation. Fig. 1 shows that control
cells grown in room air had weak NF-
B immunofluorescence. The signal
was evident primarily in the cytoplasm although there was limited fluorescence in the nuclei of some cells. By 30 min of hyperoxia, nuclear fluorescence was more prominent, and it increased over the
course of 1 day. By 24 h of hyperoxia, the cells already showed signs of swelling, and fluorescence was more intense both in the nuclei
as well as in the cytoplasm of many cells. In contrast to hyperoxia,
there was no nuclear translocation of NF-
B when cells were exposed
to concentrations of H2O2 that caused apoptosis (Fig. 1, panels E-G). By 4 h of
H2O2 treatment, the majority (~80%) of cells
had undergone apoptosis (data not shown).
Fig. 1 suggests that an increase in NF-B protein levels occurred
during hyperoxia in addition to nuclear translocation. To examine this
further, Western blots were performed. Fig.
2 shows that NF-
B levels were
increased as soon as 30 min after exposure to 95% O2, and
peak levels were achieved by 24 h. Levels remained elevated for 3 days (Fig. 2). In sharp contrast, apoptotic concentrations of
H2O2 caused no increased NF-
B protein, and
there was even a slight decrease after 2 h (Fig. 2).
To determine if elevated NF-B protein levels were correlated with
increased mRNA abundance, Northern blot analyses were performed. Fig. 3 shows that steady-state levels of
NF-
B mRNA were elevated as soon as 30 min after O2
exposure. Message levels increased over the course of 1 day and
remained elevated for 3 days. In contrast, the abundance of the message
encoding the glyceraldehyde-3-phosphate dehydrogenase was unchanged
(data not shown). Unlike hyperoxia, exposure of the culture to an
apoptotic dose of H2O2 failed to induce NF-
B
mRNA in these epithelial cells. Rather, a modest decrease was
observed after 2 h (Fig. 3).
The differential activation and expression of NF-B during different
modes of cell death imply that different signals are transduced by
hyperoxia and H2O2. We therefore investigated
whether p42 and p44 MAPKs (mitogen-activated protein kinases) were
activated. p42 and p44 MAPKs both function in a protein kinase cascade
that plays a critical role in the regulation of cell growth and
differentiation (20-23). MAPKs are activated by variety of
extracellular signals, and p42 and p44 MAPKs phosphorylate many key
regulatory proteins and transcription factors that regulate cell
proliferation. In addition, recent work showed that TNF, which
activates NF-
B, also activates the MAPK cascade (24). To study MAPK,
we used antibodies that are specific to the phosphorylated or activated form of the proteins. Fig. 4 shows that
there were no changes detected in the levels of phosphorylated p42 or
p44 at any time during exposure to hyperoxia (phosphorylated p42 is
predominant). In contrast, as soon as 10 min after incubation in an
apoptosis-inducing concentration of H2O2, there
was a significant increase in phosphorylated p42. Phosphorylated p44
was also increased, although lower in abundance. Interestingly, this
activation was transient, and the phosphorylated protein levels
decreased rapidly.
Multiple lines of evidence indicate that apoptosis results from
severe oxidative insults, and it has even been suggested that apoptosis
triggered by non-oxidative insults requires lipid peroxidation in the
dying cells (25). However, cellular oxidation also can activate
NF-B, which occurs through the redox-sensitive disassociation between NF-
B and its inhibitory protein I
B (26). Several groups have very recently reported that activation of NF-
B can prevent cell
death induced by TNF, X-irradiation, and chemotherapeutic agents
(12-14, 17). In the absence of NF-
B expression or activation, these
insults induce apoptosis in a variety of cell types. Taken together,
these observations suggest that some cells might utilize a strategy to
prevent death resulting from oxidant injury by activating NF-
B,
which presumably regulates downstream genes that salvage the cell. We
tested this notion in two models of oxidant-induced cell death by
exposing cells to lethal concentrations either of hyperoxia or
H2O2. Surprisingly, we found that despite the
rapid activation of NF-
B, hyperoxia remains lethal, indicating that NF-
B is not sufficient to protect the cells from this oxidative insult. Moreover, when apoptosis was triggered in the cells by another
oxidative insult, H2O2, NF-
B was not
induced.
The mode of cell death induced by these two oxidative insults is
different, hyperoxia caused necrosis while H2O2
caused apoptosis (11). Thus, although recent reports indicate that
apoptotic cell death is avoidable by NF-B activation, they did
not address whether NF-
B could provide protection from other modes
of cell death. Similarly, the time course of those studies was
relatively short, and the data did not bear on slower cell death that
occurs over the course of several days. Our observation that oxidative necrotic cell death can ensue despite NF-
B activation supports the
notion that strategies aimed at blocking NF-
B activation might
accelerate cell death (via apoptosis) but implies that with sufficient time the overall amount of cell death might be no different. Also, because necrosis in vivo is associated with
inflammation and apoptosis typically is not, when radiation or
chemotherapy are administered to patients, it might actually be
preferable to encourage or at least permit some degree of inflammation
to hasten the removal of dead tumor cells.
Interestingly, not only was NF-B activated, but its expression was
also induced at the protein and mRNA levels, and increasing amounts
of NF-
B were found in cells exposed to prolonged hyperoxia. This
suggests that NF-
B synthesis was also persistent. In attempting to
identify the pathway leading to NF-
B activation and induction by
hyperoxia, we found that the MAP kinase cascade was not induced, which
is distinct from other observations (24).
Non-apoptotic epithelial cell death, which is morphologically similar
to hyperoxia, occurs at lower (and perhaps more physiologically relevant) levels of oxidants than are required for the induction of
apoptosis (11). Interestingly, it is also known that non-apoptotic cell
death in the roundworm Caenorhabditis elegans, can occur in
mutants of a gene family that include deg-1, mec-4, and
mec-10. In these cases, cell death morphologically resembles
hyperoxic or low oxidant cell necrosis and is characterized by swelling and lysis of a specific group of neurons (27). Likewise, germ cells in
mes-3 worms undergo a necrosis, not apoptosis
(28). Although mec-4, encodes a subunit of a mechanosensory
ion channel, the function of these genes are not yet fully understood.
Perhaps they are steps along a pathway leading to necrosis. Taken
together with our observations, it may prove valuable to explore
pathways to alternate (non-apoptotic) modes of cell death.