From the Departments of Medicine and
§ Cell Biology, Duke University Medical Center, Durham,
North Carolina 27710, the ** Veterans Administration Geriatrics Research
and Clinical Center, Durham, North Carolina 27710, and
Laboratoire de Biochimie, Institut Louis Bugnard,
31403 Toulouse, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tumor necrosis factor- (TNF
)-induced cell
death involves a diverse array of mediators and regulators including
proteases, reactive oxygen species, the sphingolipid ceramide, and
Bcl-2. It is not known, however, if and how these components are
connected. We have previously reported that GSH inhibits, in
vitro, the neutral magnesium-dependent
sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this study,
GSH was found to reversibly inhibit the N-SMase from human mammary
carcinoma MCF7 cells. Treatment of MCF7 cells with TNF
induced a
marked decrease in the level of cellular GSH, which was accompanied by
hydrolysis of sphingomyelin and generation of ceramide. Pretreatment of
cells with GSH, GSH-methylester, or N-acetylcysteine, a
precursor of GSH biosynthesis, inhibited the TNF
-induced
sphingomyelin hydrolysis and ceramide generation as well as cell death.
Furthermore, no significant changes in GSH levels were observed in MCF7
cells treated with either bacterial SMase or ceramide, and GSH did not
protect cells from death induced by ceramide. Taken together, these
results show that GSH depletion occurs upstream of activation of
N-SMase in the TNF
signaling pathway.
TNF has been shown to activate at least two groups of caspases
involved in the initiation and "execution" phases of apoptosis. Therefore, additional studies were conducted to determine the relationship of GSH and the death proteases. Evidence is provided to
demonstrate that depletion of GSH is dependent on activity of
interleukin-1
-converting enzyme-like proteases but is upstream of
the site of action of Bcl-2 and of the execution phase caspases. Taken
together, these studies demonstrate a critical role for GSH in TNF
action and in connecting major components in the pathways leading to
cell death.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis, also known as programmed cell death, is an essential
and closely regulated process important in the development and
maintenance of multicellular organisms (1). Inducers of apoptosis
include cytokines, chemotherapeutic agents, and stress conditions.
Intracellular mediators of apoptosis include proteases of the
interleukin-1-converting enzyme/Ced-3 family (2). The cytokine tumor
necrosis factor-
(TNF
)1
induces apoptosis in target cells through binding to its cell surface
receptor, TNF
receptor 1. Recently, it has been proposed that TNF
exerts its death signal through a series of protein domain-domain
interactions; the cytoplasmic segment of TNF
receptor 1 binds to
TRADD, which in turn associates with FADD, and the latter is thought to
interact with MACH/FLICE (caspase-8) (3, 4).
Generation of oxidative stress has been proposed as a critical event
for TNF as well as other death-inducing agents in the process of
initiating their cytotoxic activity (5-9). Specifically, depletion of
glutathione (GSH), the most abundant intracellular thiol-containing
small molecule, has recently been found to either precede the onset of
apoptotic cell death induced by various agents (10-17) or render the
cells more sensitive to apoptotic agents (18-20), and it has been
suggested that depletion of GSH is a relatively early event in the
commitment to apoptosis (10). However, how this transmits cytotoxic
signals remains to be answered.
Ceramide, the product of cytokine-activated and sphingomyelinase
(SMase)-catalyzed hydrolysis of sphingomyelin (SM), is an important
regulator of apoptosis (21). SM is one of the most abundant
sphingolipid species in cell membrane with important structural and
functional properties (22, 23). Activation of SMases is believed to be
involved in cell growth, differentiation, and apoptosis induced by
cytokines, chemotherapeutic agents, and ionizing radiation (24). To
date, five types of SMase have been described, and they differ in
subcellular location, pH optimum, cation dependence, and role in cell
regulation (25). The lysosomal acid SMase has been cloned, and this
enzyme is activated in cells exposed to radiation (26), FAS (27), and
TNF (28, 29). The neutral and magnesium-dependent SMase,
although not yet cloned, has been implicated in mediating apoptosis
in cells exposed to serum starvation, FAS, TNF
, and cytosine
arabinoside (29-33). Relatively little is known about the biological
relevance of the other three SMases, namely the
zinc-dependent and lysosomal acid SMase-derived acid enzyme
(34, 35), the magnesium-independent N-SMase (36), and the alkaline
SMase (37).
We have observed that the N-SMase from the human acute lymphoblastic
leukemic Molt-4 cells is inhibited in vitro by GSH (38). This suggested to us that N-SMase may be a direct target for
transmitting at least some of the effects of GSH depletion. In the
current study, we report that GSH inhibits the TNF-induced
activation of N-SMase in MCF7 cells, and depletion of GSH induced by
TNF
involves activity of a cow pox virus cytokine modifier protein (cytokine response modifier A; CrmA)-sensitive
interleukin-1
-converting enzyme-like protease but not Bcl-2. The
implications of our results in bridging the fields of oxidative stress
and sphingolipid signaling are discussed.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
MCF7 cells (39) were cultured in RPMI 1640 (Life Technologies,
Inc.) containing 10% fetal bovine serum (Life Technologies, Inc.;
complete medium) at 37 °C in 5% CO2. For cells
transfected by Bcl-2 and its vector, hygromycin (150 µg/ml,
Calbiochem) was included in the medium, and for cells transfected by
CrmA and its vector, Geneticin (500 µg/ml, Life Technologies, Inc.)
was used. For treatment, cells were normally seeded at 5 × 105 cells/10-cm culture dish (Falcon) in 10 ml of complete
medium and grown for 48 h to 50-75% confluence. Prior to the
initiation of treatment, cells were rested in fresh complete medium
without the selection antibiotic but with 25 mM HEPES, pH
7.4. GSH was made as a 200 mM stock solution in RPMI and
added to cells 2 h before the addition of TNF. Unless otherwise
indicated, the vector control cell line for the Bcl-2-transfected MCF7
cells was used and designated as MCF7 cells.
TNF was a gift from Dr. Phil Pekana (East Carolina University,
Greenville, NC). [3H]choline chloride was purchased from
NEN Life Science Products. GSH methyl ester and YVAD were from BACHEM
(Torrance, CA). GSH and all other reagents were obtained from Sigma.
Methods
Partial Purification of N-SMase--
MCF7 cells grown to near
confluence in 175-cm2 flasks were harvested by
trypsinization, washed with ice-cold phosphate-buffered saline (PBS),
pelleted by centrifugation, frozen in a methanol-dry ice bath, and
stored at 80 °C until use. To obtain N-SMase,
detergent-solubilized membrane proteins were prepared from homogenate
from pooled cell pellets (2 × 109 cells) and resolved
on a DEAE-Sepharose column (1 × 10 cm) connected to an Amersham
Pharmacia Biotech FPLC system essentially as described (38), except
that both buffers A and B contained Triton X-100 (0.005%, w/v).
N-SMase was efficiently resolved from A-SMase in MCF7 cells (25) by the
detergent extraction step and DEAE column, such that the final N-SMase
preparation contained <1% of A-SMase activity under the assay
conditions.
N-SMase Activity Assay-- The activity of N-SMase was determined using a mixed micelle assay system as described (38). The reaction mixture contained enzyme preparation in 100 mM Tris-HCl, pH 7.4, 10 nmol of [14C]sphingomyelin (100,000 dpm), 0.1% Triton X-100, and 5 mM magnesium chloride in a final volume of 100 µl.
Measurement of Ceramide-- Cells grown in 10-cm Petri dishes were rinsed with ice-cold PBS and scraped into methanol. Cell lipids were extracted by the method of Bligh and Dyer (31). Ceramide content was determined using a modified diacylglycerol kinase assay as described previously (39).
SM Level--
Cells were seeded at 2 × 105
cells/10-cm culture dish and grown for 48 h. Then cells were
switched to fresh complete medium containing [3H]choline
(1 µCi/ml). After 48 h, cells were switched again to fresh
medium and chased for 2 h before treatment with GSH and/or TNF
as described above. The level of SM was determined following a protocol
essentially as described (40).
Measurement of GSH Level-- Cells were seeded at 2 × 105 in 6-cm Petri dishes in 4 ml of complete medium. Two days later, cells were treated with the desired agents as described above. Treated cells were detached by trypsinization, washed (three times) with ice-cold PBS, and solubilized in 150 µl of water. 5-Sulfosalicylic acid was added to a final concentration of 2%, and the supernatant was separated from the acid-precipitated proteins by centrifugation. GSH content in the supernatant was determined by the Griffith (41) modification of the Tietze's enzymatic procedure (42) as described (38). Protein content was determined by the dye binding assay using bovine serum albumin as standard (43).
Western Blot for PARP-- Cells were scraped into medium, pelleted by centrifugation, and washed (one time) with ice-cold PBS containing 1 mM phenylmethylsulfonyl fluoride. The cell pellet was resuspended in 50 µl of PBS-phenylmethylsulfonyl fluoride and solubilized in 2× SDS sample buffer. Western blot for PARP was performed as described (39, 44).
Cell Viability--
The viability of cells was determined by
their ability to exclude trypan blue. The survival of cells was
determined with the WST-1 cell proliferation reagent from Boehringer
Mannheim. Cells were seeded at 103 cells/well/200 µl of
complete medium in a 96-well culture plate. Two days later, cells were
treated in quadruplicate with TNF as described above. At the end of
treatment, WST-1 reagent was added, and after a 3-h incubation period
the absorbance was measured at 450 nm with a multiwell plate reader as
recommended by the manufacturer.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GSH Reversibly Inhibits Neutral SMase in Vitro--
Previously, we
found that GSH inhibited in vitro the N-SMase from Molt-4
leukemic cells (38). To study the role of GSH in TNF signaling, we
chose the human mammary carcinoma cell line MCF7, which is very
sensitive to TNF
(39). We partially purified N-SMase from MCF7 cells
following the procedure described for rat brain (38) and tested the
effects of GSH on N-SMase in vitro. When the enzyme was
preincubated for 5 min at 37 °C with 1-20 mM GSH
followed by incubation with substrate for 30 min, a
dose-dependent inhibition of N-SMase by GSH was observed
with a greater than 95% inhibition observed with 3 mM of
GSH (Fig. 1A). Preliminary experiments established that with the minimum preincubation time examined (1 min), GSH (3 mM) inhibited the enzyme activity
by >80% (data not shown). The inhibition was specific for GSH, since two other small thiol-containing molecules, dithiothreitol (DTT) and
-mercaptoethanol, at concentrations up to 20 mM, were
ineffective (Fig. 1A), and co-incubation of GSH with 5 or 20 mM DTT did not alter the inhibitory profile for GSH (Fig.
1B). When the N-SMase/GSH (4 mM) mixture was
diluted by 3- or 5-fold, enzyme activity was recovered by 70 and 95%,
respectively, suggesting that the inhibition was reversible (Fig.
1C). GSH did not inhibit the acidic SMase (38), although
both the N-SMase and acidic SMase have been suggested to be activated
in cells treated with TNF
(29, 33). These results demonstrate that
physiologic levels of GSH (3-10 mM) totally inhibit
N-SMase. The results also suggest that the sharp drop in cellular
levels of GSH may relieve this inhibition and cause activation of
N-SMase.
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our current study implicates GSH in regulation of a number of
TNF-induced processes. Specifically, we show that TNF
causes a
dramatic depletion of GSH, which is closely related to regulation of
neutral sphingomyelinase and activation of proteases. We show that GSH
inhibits N-SMase in vitro and provide evidence for
regulation of N-SMase in cells. Replenishment of GSH prevents
hydrolysis of SM and offers partial protection against TNF
-induced
generation of ceramide, PARP proteolysis, and cell death. Importantly,
activation of interleukin-1
-converting enzyme-like proteases, those
that are inhibited by CrmA or YVAD, is implicated in the depletion of
GSH. On the other hand, Bcl-2 does not modulate GSH depletion. Since
Bcl-2 inhibits activation of the CPP32 family of proteases (Group II;
Ref. 68) and since GSH plays a role in regulating proteolysis of PARP,
the best studied substrate of this group, these results suggest that
GSH depletion, SM hydrolysis, and ceramide accumulation (39, 50) occur
at a point upstream of activation of this group of proteases and
upstream of the site of action of Bcl-2.
Depletion of GSH has been observed in response to many inducers of
apoptosis, including TNF (51-53), FAS (11, 14), chemotherapeutic agents (54-56), viral infections (7), and glutamate (20, 57). An
important implication of this study is the prediction that those agents
that cause GSH depletion will also cause activation of N-SMase as a
direct consequence of this depletion, which would relieve the enzyme
from inhibition by GSH. Ceramide generated through this mechanism would
connect GSH depletion to a number of downstream targets known to be
modulated by ceramide, such as caspase 3/CPP32 (58), protein kinase
C
(59), and phospholipase D (60-64).
These observations also raise a number of important implications and
questions. First, how does the activation of CrmA- and YVAD-inhibitable
proteases result in depletion of GSH? Second, although this study
identifies N-SMase as a direct target regulated by GSH, it is
conceivable that the drop in GSH will affect other direct targets that
may "communicate" additional messages in response to this drop.
Third, although the results suggest an important role for GSH in
regulating N-SMase with complete inhibition of SM hydrolysis by GSH,
the effects on ceramide levels are incomplete. This raises the
possibility that ceramide is regulated by additional pathways not
involving N-SMase, such as the acidic SMase or ceramide synthase, each
of which has been implicated in ceramide formation (33, 65-67).
Finally, GSH repletion did not reverse in full the effects of TNF on
PARP proteolysis or on cell death, suggesting that additional
mechanisms are involved.
In conclusion, these results seem to connect important regulators in
TNF-induced cell death. They provide for a direct target, N-SMase,
that detects changes in the level of GSH, which may be of generalized
significance in connecting oxidative damage with sphingolipid
signaling.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Department of Defense Grant AIBS-516 (to Y. A. H.), National Institutes of Health Grants GM-43825 (to Y. A. H.) and AG-12467 (to L. M. O.), and individual National Research Service Award GM-17426 (to B. L.).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.
¶ A Terry Seelinger Fellow in Cancer.
To whom correspondence should be addressed. Present address:
Dept. of Biochemistry, Medical University of South Carolina, 171 Ashley
Ave., Charleston, SC 29425.
1
The abbreviations used are: TNF, tumor
necrosis factor-
; CrmA, cytokine response modifier A; DTT,
dithiothreitol; SM, sphingomyelin; SMase, sphingomyelinase; N-SMase,
neutral magnesium-dependent sphingomyelinase; PARP,
poly(ADP-ribose) polymerase; YVAD,
Ac-Tyr-Val-Ala-Asp-chloromethylketone; GSH-ME, GSH-methylester; PBS,
phosphate-buffered saline; NAC, N-acetylcysteine; BSO,
L-buthionine-(S,R)-sulfoximine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|