1 Department of Basic Pharmaceutical Sciences, Health Sciences Center, West Virginia University, Morgantown 26506; 2 Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505; and 3 Nelson Institute of Environmental Medicine, New York University, Tuxedo, New York 10016
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ABSTRACT |
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The APO-1/Fas
ligand (FasL) and tumor necrosis factor- (TNF-
) are two
functionally related molecules that induce apoptosis of
susceptible cells. Although the two molecules have been reported to
induce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-
, raising the
possibility that TNF-
may be involved in FasL-induced
apoptosis. Because TNF-
gene expression is under the control
of nuclear factor-
B (NF-
B), we investigated whether FasL can
induce NF-
B activation and whether such activation plays a role in
FasL-mediated cell death in macrophages. Gene transfection studies
using NF-
B-dependent reporter plasmid showed that FasL did activate
NF-
B promoter activity. Gel shift studies also revealed that FasL
mobilized the p50/p65 heterodimeric form of NF-
B. Inhibition of
NF-
B by a specific NF-
B inhibitor, caffeic acid phenylethyl
ester, or by dominant expression of the NF-
B inhibitory subunit
I
B caused an increase in FasL-induced apoptosis and a
reduction in TNF-
expression. However, neutralization of TNF-
by
specific anti-TNF-
antibody had no effect on FasL-induced
apoptosis. These results indicate that FasL-mediated cell death
in macrophages is regulated through NF-
B and is independent of
TNF-
activation, suggesting the antiapoptotic role of NF-
B
and a separate death signaling pathway mediated by FasL.
tumor necrosis factor- receptor; caspase-activated
deoxyribonuclease
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INTRODUCTION |
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THE MAINTENANCE OF CELL homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The Fas/APO-1 (CD95) and tumor necrosis factor receptor (TNF-R) are members of the TNF/nerve growth factor receptor superfamily involved in various forms of physiological and pathological cell death. The Fas and TNF-R are homologous proteins, and both have death domains in their cytoplasmic regions. These domains have the ability to initiate cell death through interaction with intracellular proteins, leading to the activation of a series of enzymes, some of which cause protein degradation (caspases; see Ref. 27) while others cause DNA cleavage [caspase-activated DNase (CAD); see Ref. 15]. The critical roles of these death receptors have been demonstrated in the mouse and in humans. For example, mice carrying naturally occurring mutations in the Fas gene (lpr mice) or in Fas ligand (FasL; gld mice) suffer from lymphoadenopathy and autoimmune diseases (10). In humans, mutations of the Fas gene that render it nonfunctional cause similar effects, producing a condition termed autoimmune lymphoproliferative syndrome (16, 37).
Increasing evidence indicates that the nuclear factor (NF)-B plays
an important role in the regulation of apoptosis by serving as
a pro- or antiapoptotic signal (1). NF-
B belongs to
a superfamily of protein dimers frequently composed of two DNA-binding
subunits, NF-
B1 (p50) and RelA (p65), which are normally
kept inactive in the cytoplasm by an attachment of the inhibitory
subunit inhibitory factor
B (I
B; see Refs. 3 and 4).
Activation of NF-
B is accomplished by phosphorylation of the I
B
by a specific I
B kinase (IKK) complex, which triggers a complete
degradation of the inhibitor (39). The activation of
NF-
B has been reported by a wide variety of
apoptosis-inducing stimuli, including oxidative stress,
cytotoxic agents, and ionizing radiations (30, 35). Additional evidence for the proapoptotic role of NF-
B is
provided by studies showing that the expression of FasL and its
induction of apoptosis in T lymphocytes require NF-
B
activation (23). Similarly, the activation of Fas death
receptor in fibroblasts is dependent on NF-
B activation
(29). The evidence that supports the antiapoptotic
role of NF-
B has mainly derived from gene knockout studies (6,
19, 24). NF-
B (p65
/
)-deficient mice die
during embryonic development through liver cell apoptosis
(6). IKK
knockout mice die as embryos and show massive
liver cell apoptosis, a response similar to that of the p65
/
knockout mice (24). Mice with an
inactivated X-linked gene encoding IKK
/NEMO, a key regulator of the
IKK complex for NF-
B activation, die at midgestation because of
massive lymphocyte apoptosis in the thymus in addition to liver
degeneration (25, 34). Thus NF-
B plays both pro- and
antiapoptotic roles in regulating apoptotic cell death
depending on cell types and physiological conditions.
Most studies investigating the role of NF-B in apoptotic cell
death have been focused on TNF-
-mediated cell death. These studies
indicate that death induced by TNF-
is negatively regulated by
NF-
B (5, 22, 41, 42). Cells that are unable to
appropriately activate NF-
B are significantly more susceptible to
TNF-
-induced apoptosis. Unlike TNF-
, the role of NF-
B
in FasL-induced apoptosis has not been consistently
demonstrated. Activation of NF-
B by Fas stimulation has been
reported to occur in some cells but not in others (7). In
TNF-
-sensitive cells, NF-
B could be activated by TNF-
but not
by Fas ligation (36). Likewise, activation of NF-
B by
TNF-
could not prevent cell death induced by FasL but by TNF-
(41). In Jurkat cells Fas causes proteolysis of NF-
B
subcomponents and prevent its activation (32). However, Fas-induced NF-
B activation has been reported (31, 32),
and this activation could protect cells against Fas-mediated cell death
(13). Interestingly, we have found that Fas stimulation can also induce TNF-
production, suggesting the possible involvement of this molecule in Fas-induced apoptosis. The objective of
this study is to clarify the involvement of NF-
B and TNF-
in
FasL-induced apoptosis and to determine whether NF-
B plays a
protective or promoting role in this process.
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MATERIALS AND METHODS |
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Cells and reagents.
The mouse macrophage cell line RAW 264.7 was obtained from the American
Type Culture Collection (Rockville, MD). The cells were maintained in
Dulbecco's modified Eagle's medium (DMEM; GIBCO Life Technologies,
Gaithersburg, MD) supplemented with 5% FBS, 2 mM glutamine, and 100 U/ml penicillin-streptomycin. Specific antibodies against NF-B p50
and p65 subunits were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA) and used in the supershift assay. The liposomal transfecting
agent LipofectAMINE was obtained from GIBCO Life Technologies.
Anti-TNF-
and anti-Fas antibodies and their isotype-matched control
antibodies were purchased from PharMingen (San Diego, CA). The NF-
B
inhibitor caffeic acid phenylethyl ester (CAPE) and recombinant FasL
(SuperFasL) were purchased from Alexis Biochemicals (San
Diego, CA). The reporter plasmid containing a luciferase gene under the
control of NF-
B promoter was a kind gift from Dr. Peter Johnson
(National Cancer Institute, Frederick, MD). The I
B plasmid was
obtained from Dr. Chuanshu Huang (Nelson Institute of Environmental
Medicine, New York University).
Detection of apoptosis. Analysis of cell apoptosis was performed by using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay kit (Boehringer Mannheim, Indianapolis, IN) and ELISA-based DNA fragmentation assay kit (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturers' instructions. For the TUNEL assay, cytospin preparations were fixed in 4% paraformaldehyde at room temperature for 5 min. After incubation with the buffer provided, the slides were immersed in terminal deoxynucleotidyl transferase (TdT) buffer, and TdT and fluorescein-dUTP were added and allowed to incubate for 60 min at 37°C. After being washed with PBS, the slides were counterstained with propidium iodide and examined under a fluorescence microscope. For ELISA assay, cells were lysed with 200 µl of lysis buffer at room temperature, and the cell lysate (20 µl) was mixed with an antibody solution (80 µl) at room temperature for 2 h. The substrate was then added after the wells were washed three times with a washing buffer. After incubation for 10 min at 37°C, the reaction was stopped, and optical density was measured using a microplate reader at a wavelength of 405 nm.
Gene transfection.
Approximately 1 × 106 cells were plated on a 12-well
plate and allowed to grow for 24 h before the transfection. The
plasmid DNA NF-B luciferase (1 µg/ml) or I
B (1 µg/ml) was
diluted in 200 µl of DMEM. The liposomal agent LipofectAMINE (12 µg/ml; GIBCO Life Technologies) was diluted in 200 µl of DMEM. The
diluted DNA and liposome samples were combined and incubated at room
temperature for 15-20 min. Cells with transfection reagents were
incubated for 4 h. Transfection medium was then replaced with
growth medium containing 10% FBS. The transfected cells were
maintained in the growth medium at 37°C for 24 h before use.
Assays of luciferase activity and TNF- protein expression.
Luciferase activity was measured by enzyme-dependent light production
using a luciferase assay kit (Promega, Madison, WI). After each
experiment, cells were washed and incubated at room temperature for 10 min in 250 µl of lysis buffer (Promega). Ten-microliter samples were
then taken and loaded in an automated luminometer (Bio-Rad, Hercules,
CA). At the time of measurement, 100 µl of luciferase substrate were
automatically injected in each sample, and total luminescence was
measured over a 20-s time interval. Output is quantitated as relative
light units per microgram protein of the sample. For analysis of
TNF-
protein, cell-free supernatants were used. TNF-
levels were
determined using a TNF-
ELISA kit (R&D System, Minneapolis, MN)
according to the manufacturer's instructions.
Flow cytometry. Cells were harvested and suspended in PBS containing 1% BSA. They were fixed with ice-cold 4% formaldehyde for 15 min and stained with a rabbit anti-Fas antibody or isotype-matched control antibody, followed by phycoerythrine-conjugated rat anti-rabbit secondary antibody. Subsequently, the cells were subjected to flow cytometric analysis with a gate set for examining a total of 104 cells.
Electrophoretic mobility shift assay.
To detect NF-B binding activity, nuclear protein extracts were first
prepared as follows: cells were treated with 500 µl of lysis buffer
[50 mM KCl, 0.5% Nonidet P-40, 25 mM HEPES, 1 mM phenylmethylsulfonyl
fluoride (PMSF), 10 mg/ml leupeptin, 20 µl/ml aprotinin, and 100 mM
dithiothreitol (DTT)] on ice for 4 min. Nuclei were pelletted by
centrifugation at 14,000 rpm for 1 min and were resuspended in 300 µl
of extraction buffer (500 mM KCl, 10% glycerol, 25 mM HEPES, 1 mM
PMSF, 1 µl/ml leupeptin, 20 µg/ml aprotinin, and 100 µM DTT).
After centrifugation at 14,000 rpm for 5 min, the supernatant was
harvested and stored at
70°C. The protein concentration was
determined using the bicinchoninic acid protein assay reagent (Pierce,
Rockford, IL).
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RESULTS |
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FasL induces apoptotic cell death in macrophage RAW cells.
Apoptosis triggered by Fas/FasL signaling is an important
process in the homeostasis of the immune system (8).
Macrophages are key cellular effectors of the immune system; however,
their regulation of apoptosis induction by Fas/FasL is largely
unknown. In the present study, we investigated the induction of
apoptosis by FasL in macrophage RAW 264.7 cells. Treatment of
the cells with FasL (0-400 ng/ml) caused a dose-dependent increase
in the level of apoptosis, as analyzed by DNA fragmentation
enzyme-linked immunosorbent assay (ELISA; Fig.
1A). Peak response time for
the apoptosis induction was ~16 h posttreatment (Fig.
1B). Morphological analysis of cell apoptosis by
TUNEL assay showed an increasing number of apoptotic cells (green
fluorescent signals) with time after FasL treatment (Fig. 1,
D-F). In contrast, no or very few apoptotic cells were
observed in the control samples (Fig. 1C). These results
indicate that FasL was able to induce apoptosis in macrophage
RAW cells.
|
Fas expression in macrophage RAW cells.
Fas expression has been reported in a variety of cells from different
tissues (43, 44). To determine whether Fas is expressed in
RAW cells, we performed flow cytometric analysis of Fas expression. The
results indicated that cells stained with the anti-Fas antibody showed
a strong positive signal (Fig.
2B), whereas those stained with the control isotype-matched antibody showed a weak signal (Fig.
2A). These results indicated the expression of Fas in RAW cells.
|
FasL-induced NF-B activation.
The nuclear transcription factor NF-
B has been shown to play a role
in regulating Fas-induced apoptosis in some cells (13, 31, 33) but not in others (7, 36, 41). To
investigate the role of this transcription factor in RAW cell
apoptosis induced by FasL, electrophoretic gel shift and gene
transfection studies were carried out. The results from the gel shift
study showed that FasL was able to induce DNA binding activity of
NF-
B at the concentration shown to induce cell apoptosis
(200 ng/ml; Fig. 3A,
lanes 1 and 2). To determine the specificity of
NF-
B binding in this assay, a nonlabeled NF-
B probe was used as
competitor for NF-
B binding. Figure 3A, lane
3, shows that the nonlabeled probe was able to compete for the
binding, whereas a nonspecific activator protein-1 probe could not
(lane 4). Supershift assays using antibodies
specific to the p50 and p65 subunits of NF-
B also showed a band
shift of the NF-
B complexes (lanes 5 and 6). Together, these results indicated the specificity of NF-
B binding and the formation of p65/p50 and p50/p50 complexes in FasL-treated cells.
|
FasL-induced TNF- expression and its effect on
apoptosis.
NF-
B has been shown to play an essential role in regulating TNF-
expression (11, 47). Because our results showed that FasL
was able to induce NF-
B activation, we therefore investigated whether FasL could induce TNF-
expression in RAW cells. Treatment of
the cells with FasL caused a strong induction in TNF-
protein expression, as analyzed by ELISA (Fig.
4A). This effect was both dose
and time dependent with a peak response time of ~8 h (Fig. 4B). Gene transfection assay with the use of a
TNF-luciferase reporter plasmid containing NF-
B-binding sites
similarly showed the activation of the TNF-
gene promoter by the
FasL treatment (results not shown). The observation that FasL-induced
TNF-
expression raised the possibility that TNF-
may be involved
in FasL-mediated cell death. To test this possibility, cells were
treated with FasL in the presence or absence of neutralizing
anti-TNF-
antibody or control isotype-matched antibody. Figure
4C shows that treatment of the cells with either the
anti-TNF-
or control antibody had no significant effect on
FasL-induced apoptosis. Varying the concentration of the
antibodies from 1 to 500 ng/ml gave similar results. These results
indicated that TNF-
was not involved in FasL-induced apoptosis in RAW cells. To further confirm the observed absense of effect of TNF-
, we directly exposed the cells to purified TNF-
and measured its effect on cell apoptosis. Figure
4D shows that the exogenously administered TNF-
had no
effect on cell apoptosis when used at the concentration range
of 0.1-10 ng/ml. This concentration range was used on the basis of
the TNF-
produced during FasL treatment (Fig. 4A). These
results confirmed the finding from antibody experiments and indicated
that TNF-
was not involved in FasL-induced apoptosis.
|
Role of NF-B in FasL-induced apoptosis.
To investigate the role of NF-
B in FasL-induced apoptosis,
cells were treated with a specific NF-
B inhibitor, CAPE, or
transfected with a dominant form of the NF-
B, I
B, and their
effects on FasL-induced apoptosis were examined. Figure
5A shows that treatment of the cells with CAPE caused an inhibition of FasL-induced NF-
B
activation. Such treatment also resulted in an increase in cell
apoptosis induced by FasL (Fig. 5B). CAPE by itself
had a partial apoptosis-inducing effect on the cells,
suggesting the role of NF-
B in the normal maintenance of cell
apoptosis. Morphological analysis of the cells by TUNEL (Fig.
5, C-F) similarly indicated the antiapoptotic role of
NF-
B and its inhibition by CAPE.
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DISCUSSION |
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FasL binding to the Fas receptor initiates apoptosis in a
variety of cell types. However, not all cell types that express Fas are
susceptible to Fas-induced apoptosis (28). For
example, cells of the lymphoid origin need to be stimulated to become
sensitive to Fas-induced cell death (2, 45). Such
activation is required to increase Fas expression and consequently its
competency to deliver the death signal. In this study, we have shown
that the macrophage RAW 264.7 cells constitutively and abundantly
express a functional Fas receptor. Activation of the cells by FasL
induces dose- and time-dependent apoptosis (Fig. 1). Such
induction is also associated with NF-B activation, the process that
was shown to negatively regulate the FasL-induced apoptosis.
This notion is supported by the following observations: 1)
FasL increases the DNA-binding activity of NF-
B and mobilizes its
p50/p65 heterodimeric form (Fig. 2A), 2) it also
increases NF-
B promoter activity of the luciferase reporter gene
(Fig. 2B), and 3) inhibition of NF-
B by the
specific NF-
B inhibitor CAPE or by dominant expression of I
B
makes the cells more susceptible to FasL-induced apoptosis (Figs. 5 and 6).
The observed activation of NF-B and its suppressive effect on
FasL-induced apoptosis are consistent with previous findings (13, 31, 33) but are contradictory to others (7, 36, 41). Because of the diverse functions of different cell types and their variable susceptibilities to apoptosis induction, it is likely that the observed discrepancies between these test results are cell type specific. Some cell types express Fas, whereas others do
not (14, 17). Additionally, some Fas-bearing cells need to
be stimulated, as described above, to become sensitive to Fas-induced cell death. In sensitive cells, the activation of NF-
B should lead
to an increased expression of NF-
B-dependent genes. One of the
prototype genes that is under the dominant control of NF-
B is the
TNF-
gene (11, 47). TNF-
is produced principally by
macrophages; therefore, activation of NF-
B by FasL should lead to an
increased expression of TNF-
. Our results showed that activation of
the cells by FasL did indeed result in an increase in TNF-
expression (Fig. 4); the effect was shown to be dependent on NF-
B
activation, since inhibition of NF-
B by I
B inhibited such
expression (Fig. 6B).
Because TNF- is known to be a positive regulator of NF-
B, its
induction during FasL stimulation suggests its possible involvement in
NF-
B activation. Our results, however, showed that NF-
B
activation occurred relatively early and peaked at ~2 h (Fig.
3A), whereas TNF-
expression occurred at later times and
peaked at ~8 h (Fig. 4B). These results, along with the
observed low level of TNF-
expression relative to the level of FasL
in the system, suggest that the activation of NF-
B during FasL
treatment is mediated primarily by FasL. The mechanism by which FasL
induces NF-
B activation is unclear, but previous studies suggest
that reactive oxygen species (ROS) may be involved (12, 18, 21,
40). Gulbins et al. (18) implicated superoxide
anion as a functional mediator of Fas-induced cell death in Jurkat
cells, whereas others (12, 21, 40) suggested the role of
multiple ROS in various cell systems. However, Hug et al.
(20) found no requirement of ROS in L929 murine
fibroblasts stably expressing Fas. Because different cell types produce
varying levels of ROS, their susceptibility to ROS-mediated NF-
B
activation is likely to be different. Supporting this notion is the
observation that the ROS-producing macrophage is highly responsive to
NF-
B activation, as demonstrated in this study.
Because TNF- is also known to be an apoptosis inducer, its
activation during FasL treatment suggests its possible involvement in
FasL-induced apoptosis. Furthermore, because Fas and TNF-R share common structural homologies and the activation of Fas, like
TNF-
(33), causes mobilization of the p50/p65
heterodimer, it is therefore possible that the two ligands may mediate
the death signal via a common signaling pathway. Using neutralizing anti-TNF-
antibody, we showed that inhibition of TNF-
by the antibody had no effect on FasL-induced cell death (Fig. 4C).
Furthermore, direct exposure of the cells to TNF-
at the
concentrations shown to be produced during FasL treatment failed to
induce cell apoptosis (Fig. 4D). These results
demonstrated that TNF-
was not involved in FasL-induced
apoptosis under the experimental conditions. The inability of
TNF-
to induce apoptosis may be a result of its relatively
low level of expression during FasL stimulation. The activation of
NF-
B by FasL may also provide a survival signal that suppresses the
potential apoptosis-inducing effect of TNF-
. These results
are consistent with previous findings (4, 26, 36) and
suggest that apoptosis mediated by FasL occurs via a separate
signaling pathway independent of TNF-
. This finding is further
supported by previous observations that FasL does not bind to TNF-R and
that apoptosis induced by FasL is mediated by the Fas receptor
(38).
In conclusion, we have shown that FasL can induce apoptosis of
macrophage RAW 264.7 cells and activate the NF-B p50/p65 complex. Such activation negatively regulates FasL-induced cell death, supporting the antiapoptotic role of NF-
B in this cell system. NF-
B may mediate its antiapoptotic effect through the activation of protective proteins such as cIAP, TRAF, and IEX-1L, which has been
previously reported (9, 42, 46). FasL activation
of RAW cells is also associated with an increased expression of
TNF-
, the process that is dependent on NF-
B activation. Despite
the structural similarities between FasL and TNF-
, and their
receptors, FasL-induced apoptosis is independent of TNF-
,
suggesting separate death signaling pathways. The ability of FasL to
activate NF-
B, a key transcription factor of various important
genes, suggests that this molecule may serve a much broader range of
biological functions in addition to its role as an apoptosis inducer.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-62959.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y. Rojanasakul, West Virginia Univ. Health Sciences Center, Dept. of Basic Pharmaceutical Sciences, P.O. Box 9530, Morgantown, WV 26506 (E-mail: yrojanasakul{at}hsc.wvu.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.
May 29, 2002;10.1152/ajpcell.00045.2002
Received 29 January 2002; accepted in final form 25 April 2002.
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