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J. Biol. Chem., Vol. 277, Issue 43, 41128-41139, October 25, 2002
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,¶,
,**,,¶¶, and
From the Departments of Biochemistry and Molecular
Biology, §§ Medicine, and
Pathology and Laboratory Medicine, Medical
University of South Carolina, Charleston, South Carolina 29425 and the
§ Divisions of Discovery Chemistry and Biology,
GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Received for publication, July 8, 2002, and in revised form, July 31, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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A high throughput screen for neutral,
magnesium-dependent sphingomyelinase (SMase) was performed.
One inhibitor discovered in the screen, GW4869, functioned as a
noncompetitive inhibitor of the enzyme in vitro with an
IC50 of 1 µM. It did not inhibit acid SMase
at up to at least 150 µM. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 µM) partially inhibited TNF-induced sphingomyelin (SM)
hydrolysis, and 20 µM of the compound was protected
completely from the loss of SM. The addition of 10-20 µM
GW4869 completely inhibited the initial accumulation of ceramide,
whereas this effect was partially lost at later time points (24 h).
These data therefore support the inhibitory action of GW4869 on N-SMase
not only in vitro but also in a cellular model. The
addition of GW4869 at both 10 and 20 µM did not modify
cellular glutathione levels in response to TNF, suggesting that the
action of GW4869 occurred downstream of the drop in glutathione, which
was shown previously to occur upstream of the activation of
N-SMase. Further, whereas TNF treatment also caused a 75% increase of
de novo synthesized ceramide after 20 h of incubation,
GW4869, at either 10 or 20 µM, had no effect on this
pathway of ceramide generation. In addition, GW4869 did not
significantly impair TNF-induced NF- Programmed cell death is a necessary requirement during
development, and an altered regulation of this process is also thought to contribute to the occurrence of serious illnesses such as cancer and
neurodegenerative diseases. Significant progress has been made in the
identification of crucial events that contribute to defining this
process (mitochondrial dysfunction, chromatin fragmentation, and
membrane blebbing) as well as in the identification of molecular mechanisms and effectors that regulate and/or execute these events (exposure of phosphatidylserine on the plasma membrane, depolarization of the outer mitochondrial membrane, formation of permeability transition pores in the mitochondrial membrane, cytochrome c
release, caspase activation, and alteration of calcium
homeostasis, Bcl2 family members, apoptosis-inducing factor,
apoptosis activating factor-1, Smac/Diablo, IAPs, and others).
The sphingolipid ceramide has been shown to induce cellular features
characteristic of programmed cell death (membrane blebbing, mitochondrial dysfunction, and nuclear fragmentation). Moreover, many
agents known to cause apoptosis (such as
TNF,1 CD95 cross-linking,
daunorubicin, heat stress, growth factor withdrawal, UV-B and
Two main routes have been defined for the generation of ceramide:
hydrolysis of sphingomyelin and de novo biosynthesis. The first occurs by the action of sphingomyelinases (SMases), which operate
at different pH optima (acid or neutral SMase) and have different metal
requirements (magnesium dependence) (2, 4). Activation of acid
sphingomyelinase (A-SMase) has been observed after treatment with UV-A
radiation (5) and stimulation of the p75 neurotropin receptor (6), CD28
(7), TNF receptor, and CD95 (8), although some of these latter
conclusions have been recently questioned (9, 10). Neutral SMase
(N-SMase) activation has been also observed after stimulation of the
p75 neurotropin receptor (11), after ligation of CD95 and TNF receptor (12, 13), and after irradiation (14) but also upon heat stress and
serum starvation (15), treatment with vitamin D (16), and CD40 ligation
(17). The de novo pathway is regulated primarily by the
action of serine palmitoyltransferase, the first enzyme along the
biosynthetic pathway. Accumulation of ceramide via de novo
biosynthesis was originally shown after treatment with retinoic acid in
GH4C1 (18) and then with daunorubicin in P388 and U937 cells (19),
angiotensin II stimulation of PC12W cells (20), etoposide treatment of
Molt-4 cells (21), IgM-induced receptor cross-linking in Ramos B cells
(22), loading of palmitate in beta cells (23), and TNF/cycloheximide
treatment of bovine cerebral endothelial cells (24).
The study of the de novo pathway has benefited tremendously
from the availability of highly specific inhibitors: fumonisin B1 for
ceramide synthase (25) and myriocin/ISP1 for serine
palmitoyltransferase (26). On the other hand, very few tools have been
available to dissect the neutral sphingomyelinase pathway. One
molecule, scyphostatin, has been previously described to exert
inhibitory activity versus a N-SMase (27, 28), and it has
been used to implicate N-SMase in the outgrowth process of hippocampal
neurons in response to nerve growth factor (29). We set out to develop specific inhibitors of N-SMase and to determine the role of N-SMase in
apoptosis and how it interacts with other key regulators of apoptosis.
A high throughput assay was developed for N-SMase, and we successfully
identified a molecule, GW4869 (Fig. 1), that exhibited significant and
specific inhibitory activity on N-SMase. We provide evidence of the
specificity of GW4869 in vitro. Next, the effects of this
inhibitor were determined in MCF7 breast cancer cells treated with TNF,
which has emerged as one of the best characterized models of
cytokine-induced cell death and of ceramide function. It has been
previously shown that TNF induces activation of N-SMase in these cells
and that this activation is a consequence of the drop of glutathione
that follows the activation of the death receptor and caspase 8 (13).
Also, this activation couples to processing of effector caspases
(30).
Using this newly characterized inhibitor, we were able to confirm
N-SMase activation upon TNF treatment of MCF7 and its effects on
effector caspases and cell death. Second, we provide evidence for the
first time of the participation of N-SMase in the processes that lead
to cytochrome c release and mitochondrial dysfunction, indicating the requirement for N-SMase activation as a necessary step
for the complete development of the cytotoxic program induced by
TNF.
Materials--
RPMI 1640 medium was from Invitrogen.
Fetal bovine serum (FBS) was from Summit Technology. TNF was from
Peprotech (Rocky Hill, NJ). [ In Vitro Sphingomyelinase Activity Assays--
Partially
purified and delipidated rat brain neutral sphingomyelinase was
prepared and assayed as described (31) with variable amounts of SM or
PS incorporated into the assay via solubilization into Triton X-100
micelles. Human acid sphingomyelinase, overexpressed in SF-9 insect
cells using a baculoviral system, was similarly assayed in a final
reaction volume of 50 µl containing 100 mM sodium acetate
(pH 5), 0.1 mM zinc acetate, 0.13% Triton X-100 and
lacking PS or dithiothreitol. The reaction was allowed to proceed for
10, 30, and 150 min at room temperature. At the appropriate time, the
reaction was quenched with 75 µl of 5% bovine serum albumin followed
by 75 µl of 8% trichloroacetic acid. After centrifugation, 100 µl
of supernatant were counted for 14C with 150 µl of
Optiphase Supermix. All rate measurements obtained were in the linear
range and represented conversion of less than 20% of substrate to product.
Partial Purification of the Lyso-PAF Phospholipase C
(PLC)--
HEK293, expressing the lyso-PAF PLC (32), were lysed in 25 mM Tris (pH 7.4), 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 2 µg/ml each of chymostatin,
leupeptin, antipain, and pepstatin A in the absence of detergents, and
the postnuclear extract was centrifuged for 1 h at 100,000 × g at 4 °C. The pellet, containing the total membranes,
was resuspended in lysis buffer containing 0.1% Triton X-100 and
incubated on a rotating platform for 1 h at 4 °C. The
suspension was then centrifuged for 1h at 100,000 × g
at 4 °C, and the supernatant was applied to a HiTrap Q column equilibrated with 20 mM Tris-HCl, pH 7.4, 0.1% Triton
X-100, 1 mM EGTA, 1 mM EDTA. The lyso-PAF PLC
was then eluted with a linear salt gradient (1.5 M NaCl).
Fractions were collected, N-SMase activity was measured as previously
indicated (33), and the protein level was determined by Western blot
analysis using anti-His6 monoclonal antibodies against the
histidine-FLAG of the protein. The fraction containing peak activity
(which also showed most of the protein by western) was used for
determining the effect of GW4869 in vitro.
Specificity of GW4869 on Enzyme Inhibition--
Partially
purified rat brain N-SMase and lyso-PAF PLC were incubated in the
absence or presence of GW4869 and PS (100 µM), and SM
hydrolysis was determined as previously described (30). In the case of
Bacillus cereus N-SMase (Sigma), PS was not included in the
reaction mixture, since it does not affect the bacterial enzymatic
activity. B. cereus phosphatidylcholine-PLC (Sigma) was
incubated in the presence or absence of GW4869 in a reaction mixture
containing 100 mM Tris, pH 7.2, 25% glycerol, 20 mM p-nitrophenyl/phosphorylcholine, and
production of p-nitrophenol was quantified
spectrophotometrically at 410 nm. Protein phosphatase 2A from bovine
kidney (Calbiochem) was incubated in the presence or absence of GW4869
in buffer containing 50 mM Tris, pH 7.4, 1 mM
dithiothreitol, 100 µM MnCl2, and 20% glycerol, and phosphatase activity was measured as described by Jones
and Hannun (34). In all assays, 10 µM GW4869 was used, and 30-min preincubation with the enzymes preceded the addition of the substrate.
Cell Culture and GW4869 Treatment--
MCF7 human breast cancer
cells were routinely cultured in RPMI 1640 containing 10% FBS at
37 °C in 5% CO2. Unless otherwise indicated, for
treatment, cells were seeded at 1.7 × 106 cells/10-cm culture
dish in 10 ml of complete medium; after 24 h, the medium was
replaced with 7 ml of RPMI 1640 containing 2% FBS and 25 mM Hepes, pH 7.5, and the cells were rested for 2 h
prior to treatment. GW4869 was routinely stored at Sphingomyelin Measurement--
Cells were seeded at 0.1 × 106 cells/10-cm dish in 8 ml of complete growth medium.
After 48 h, the cells were labeled with [methyl-3H]choline chloride (1 µCi/ml final
concentration in 10 ml of growth medium/plate). After ~60 h, the
cells were chased with 10 ml of complete medium for 90 min. Then the
cells were washed once with 5 ml of PBS, and 7 ml of medium containing
2% FBS and 25 mM Hepes, pH 7.5, were added. After resting
the cells for approximately 1 h, preincubation for 30 min with
GW4869 was started, and TNF treatment followed. At the appropriate time
points, the medium from each plate was collected, and the cells were
washed once with 2 ml of ice-cold PBS. Cells were scraped on ice in 2 ml of PBS, and each plate was washed with an additional 2 ml of PBS. Cells and washes were pooled with the medium and centrifuged for 5 min
at 2000 × g (4 °C). The cell pellets were stored at
Measurements of Mass Levels of Ceramide--
Cells were
harvested in methanol, and lipids were extracted by the method of Bligh
and Dyer (36). The chloroform organic phase was divided into aliquots
(in duplicates) and dried down for ceramide and phosphate measurements.
Ceramide levels were evaluated using the Escherichia coli
diacylglycerol kinase assay. Ceramide was quantitated by using external
standards and normalized to phosphate content (37).
Measurement of Ceramide Generated by de Novo
Biosynthesis--
Cells were seeded at 1.7 × 106
cells/plate in complete growth medium. After 24 h, the medium was
replaced with 6 ml of RPMI 1640 containing 2% FBS and 25 mM Hepes, pH 7.5. Right before the addition of GW4869,
[3H]palmitate was added to the cells in 1 ml of the same
medium to a final activity of 1 µCi/ml. After ~21 h of treatment
with TNF, the medium was collected, and the plates were washed once with PBS that was combined with the medium and centrifuged at 2000 × g for 5 min at 4 °C to collect floating cells. Cells
were scraped off the plate with methanol and combined with the
floaters. Lipids were extracted by the method of Bligh and Dyer (36). One ml of the organic phase was used for ceramide determination by
separation of lipids through thin layer chromatography
(chloroform, methanol, 4.2 N ammonium hydroxide;
4:1:0.1; v/v/v), and 0.35 ml in duplicates were used for determination
of inorganic phosphate used to normalize the ceramide values. The
ceramide band was identified by comparison with an authentic standard,
and radioactivity was quantified in a scintillation counter.
Electrophoretic Mobility Shift Assay of NF- Immunocytochemistry of NF- Glutathione Measurements--
At the indicated time points,
adherent cells were collected by trypsinization and combined with
floaters in the medium. After centrifugation, cells were washed twice
with ice-cold PBS and resuspended in 0.4 ml of ice-cold
double-distilled H2O. The following steps were performed at
4 °C using a modified procedure from Tietze F (39, 40). Briefly, 0.1 ml of lysate were used for protein determination using the Bio-Rad
assay, and the remaining 0.3 ml of lysate were added to 75 µl of 10%
(v/v) 5-sulfosalicylic acid, mixed, and incubated on ice for 10 min to
allow protein precipitation. Samples were then centrifuged, and
supernatants were stored at In Vitro Caspase Activity Assay--
Caspase activity was
analyzed with the ApoAlert CPP32/caspase 3 assay kit according to the
manufacturer's protocol (CLONTECH, Palo Alto, CA).
At the indicated time points, adherent cells were collected by
trypsinization and combined with floaters in the medium. After
centrifugation, cells were washed twice with ice-cold PBS and stored as
a pellet at MTT Assay--
5 × 103 cells/well were seeded in a 96-well
plate in 75 µl of RPMI containing 2% FBS and 25 mM
Hepes, pH 7.5. After 24 h, first GW4869 was added in 15 µl of
medium/well and incubated for 30 min and then TNF was added in 10 µl/well (total volume of 100 µl/well). At the indicated time
points, 25 µl of MTT stock solution (5 mg/ml in PBS) were added to
each well and incubated at 37 °C in 5% CO2 for 3 h. Subsequently, cells were solubilized by the addition of 100 µl of
lysis buffer (20% SDS (w/v), 50%
N,N-dimethylformamide (v/v), 0.8% acetic acid
(v/v), pH 4.6-4.8) to each well. The production of the formazan dye
was quantitated by measuring the OD at 595 nm with a multiwell plate
reader. We have noticed that the efficacy of TNF to induce
morphological changes was significantly slower in experiments carried
out in the 96-well plates (as for the MTT) compared with those
performed in 10-cm Petri dishes (all other experiments).
Preparation of Cytosolic Extracts--
At the indicated time
points, plates were washed once with ice-cold PBS, and floaters in the
medium and from the washes were collected by centrifugation. Adherent
cells were scraped in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EGTA, 2 mM EDTA, 10 mM sodium orthovanadate, 20 mM sodium fluoride,
1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 0.5% Nonidet P-40, and 10 µg/ml each of
chymotrypsin, leupeptin, aprotinin, and pepstatin). After combining
with floaters, cells were incubated on ice for 10 min. Lysates were
then centrifuged at 1000 × g for 10 min to remove unbroken cells and nuclei, and supernatants were centrifuged at 100,000 × g for 1 h to obtain cytosolic fractions
that were stored at Western Blotting--
Equal amounts of protein, usually 80 µg,
were resolved by 7, 12, and 15% SDS-PAGE for analysis of PARP and
caspases 6 and 9, respectively. After transfer to a nitrocellulose
membrane, the proteins were incubated with anti-PARP rabbit polyclonal
IgG (1:2500; Santa Cruz Biotechnology), anti-human caspase 6 monoclonal IgG (2 µg/ml; Pharmingen), or anti-caspase 9 rabbit polyclonal IgG
(1:500; Santa Cruz Biotechnology) and secondary anti-rabbit (1:4000)
and anti-mouse (1:5000) antibodies (Santa Cruz Biotechnology). The
signal was visualized by enhanced chemiluminescence (Amersham Biosciences).
Electron Microscopy Analysis--
Cells were seeded at a
concentration of 0.4 × 106 cells/25-cm2
flask in growth medium. After 48 h, medium was changed with
RPMI 1640 containing 2% FBS and 25 mM Hepes, pH 7.5, and
cells were rested for 1.5 h. Cells were pretreated with GW4869 and
treated with TNF in a total volume of 4 ml. After 14 h, cells were
fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer for
30 min and rinsed overnight in cacodylate buffer with 7% sucrose.
Cells were then dehydrated in graded ethyl alcohol as follows: 5 min in
50% ethyl alcohol, 5 min in 70% ethyl alcohol, 5 min in 95% ethyl alcohol, 10 min in 100% ethyl alcohol, 10 min in 100% ethyl alcohol. Samples were infiltrated with a 1:1 solution of 100% ethyl alcohol and
Embed 812 embedding resin for 30 min and embedded (in the flask) with
Embed 812 embedding resin. Samples were polymerized for 24 h and
capped in a 60 °C oven, and polymerization was allowed to continue
for an additional 24 h (caps off). The plastic flask was broken
away from the hardened resin, and representative blocks were sawed out
using a Sears electric scroll saw. The blocks were re-embedded for
orientation and polymerized for 24 h at 60 °C. Thick sections
were cut using the Riechert Ultramicrotome. These 0.5-µm
sections were stained with toluidine blue and examined with the light
microscope. Those blocks containing the appropriate monolayer of
cells were chosen for thin sectioning. Thin sections were cut using the
Riechert Ultramicrotome Ultracut E. These 70-nm thin sections
were picked up on copper grids and double-stained with uranyl acetate
and lead citrate. Thin sections were viewed in the JEOL 1210 transmission electron microscope, and representative digital images
were saved.
In Vitro Studies
GW4869 was discovered during a high throughput screen with a
preparation of delipidated rat brain neutral SMase. The compound structure is shown in Fig. 1, and
inhibition of N-SMase by the compound is shown in Fig.
2. GW4869 acted as a noncompetitive inhibitor with the substrate sphingomyelin (Fig. 2A), and
the IC50 for the interaction is 1 µM (Fig.
2B). The Km for sphingomyelin under these
conditions was found to be 13 µM (0.4 mol % at 0.2%
Triton X-100, 0.02% PS, and 10 mM MgCl2). The
compound showed competitive characteristics toward the activator PS
(Fig. 3A); however, at high PS
concentrations, the inhibition was more complex, since at these high PS
levels, total inhibition was not observed under standard assay
conditions. Since PS, an anionic lipid, could bind Mg2+, we
evaluated the effects of varied Mg2+ levels under high PS
conditions. Fig. 3B shows that at higher Mg2+
levels, inhibition again became complete. The effect of the compound on
the A-SMase was also determined. At up to 150 µM, GW4869
did not inhibit the cloned human A-SMase (Fig.
4). Additionally, the effect of GW4869 on
other hydrolytic enzymatic activities was also tested. As reported in
Table I, only rat brain and bacterial neutral SMases were efficiently inhibited by GW4869, whereas no inhibitory activity was observed for the bacterial
phosphatidylcholine-specific PLC, and minimal inhibition was observed
for the mammalian lyso-PAF PLC, an enzyme that also hydrolyzes SM
in vitro. Finally, minor inhibitory effects were observed
for bovine protein phosphatase 2A activity, a target for ceramide.
Overall, the results show that GW4869 is a selective inhibitor of
N-SMase, especially among enzymes known to act on SM.
B translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent
manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue
uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and
caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that
N-SMase activation is a necessary step for the full development of the
cytotoxic program induced by TNF.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-radiation, bacterial infections, and others) have also been found
to increase the intracellular levels of ceramide, with emerging
evidence suggesting an important role for ceramide in
regulating/mediating the apoptotic response to these agents (for
reviews, see Refs. 1-3). A major current challenge is to define the
role of the pathway promoted by ceramide and integrate it with other
molecular mechanisms that lead to cell death.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP,
[methyl-3H]choline chloride, and
EN3HANCETM spray were from PerkinElmer Life
Sciences.
[choline-methyl-14C]SM was
provided by Alicja Bielawska (Medical University of South Carolina,
Charleston, SC). SM and PS were from Avanti Polar Lipids, Inc. Silica
Gel 60 thin layer chromatography plates were from Whatman.
Scintillation mixture Safety Solve was from Research Products
International. Poly(dI-dC) and poly(dN6) were from Amersham Biosciences. Rabbit anti-p65 NF-B antibodies were from Rockland; monoclonal anti-cytochrome c and anti-human caspase 6 antibodies were from Becton Dickinson Co.; and rabbit anti-PARP,
anti-mouse IgG-horseradish peroxidase, and anti-rabbit IgG-horseradish
peroxidase were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
80 °C as a 1.5 mM stock suspension in Me2SO. Right
before use, the suspension was solubilized by the addition of 5%
methane sulfonic acid (MSA) (2.5 µl of 5% MSA in sterile
double-distilled H2O were added to 50 µl of GW4869 stock
suspension; therefore, the concentration of the GW4869 stock solution
at the time of the experiments was 1.43 mM). The suspension was mixed and warmed up at 37 °C until clear. Cells were
preincubated with the inhibitor for 30 min prior to treatment with TNF.
Control cells were treated with Me2SO containing 5% MSA,
similarly to the samples receiving the GW4869 solution. When different
doses of GW4869 were tested, amounts of vehicle solution were added in
order to equal the volume of GW4869 used for the highest dose.
80 °C. On the day of the measurement, the pellets were resuspended in 600 µl of double-distilled H2O by vortexing and
sonication. Aliquots of cell lysates were used for protein
determination, and 250 µl in duplicate were used for SM determination
as described by Andrieu et al. (35).
B--
The assay
was performed as previously described (38). Briefly, after treatments,
cells were washed and harvested by scraping in PBS. The pellet was
resuspended in 400 µl of lysis buffer (10 mM Hepes, pH
7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride) and incubated for 10 min on ice. Just
prior to centrifugation, 20 µl of 10% Nonidet P-40 was added, and
the suspension was mixed by pipetting up and down three times. Nuclei
were pelleted by microcentrifugation at 1300 × g for
10 min. The supernatant was removed, and the nuclei were resuspended in
20 µl of extraction buffer (20 mM Hepes (pH 7.9), 0.4 M NaCl, 25% (v/v) glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). The suspension was mixed gently for 30 min and pelleted at 13,000 × g for 15 min. The supernatant was flash-frozen and stored at 80 °C. Protein
concentrations were determined using the Bio-Rad assay. Nuclear
extracts (10 µg) were incubated in HDKE buffer (20 mM
Hepes, pH 7.9, 50 mM KCl, 5% (v/v) glycerol, 1 mM EDTA, 5 mM dithiothreitol) containing 1 µg
of poly(dI-dC), 1 µg of poly(dN6), and 10 µg of bovine serum albumin. One µl of radiolabeled oligonucleotide probe
(60,000-100,000 cpm) was added to each reaction and incubated at room
temperature for 20 min. The reaction was terminated by the addition of
6 µl of 15% Ficoll solution containing indicator dyes (bromphenol
blue and xylene cyanol). Equal amounts (20 µl) of reaction mixture were loaded on a 5% nondenaturing polyacrylamide gel in 1× TBE and
run at 200 V. Gels were placed onto Whatman filter paper, dried, and
autoradiographed. The specificity of NF-B activation upon TNF
stimulation in the absence and in the presence of GW6948 was assayed as
previously described (34).
B and Cytochrome c and Nuclear
Staining--
For immunocytochemical analysis, cells were plated at a
density of 1.7 × 106 cells/100-mm plate that
contained 22-mm glass coverslips. Following treatment, cells were fixed
for 15 min in 4% paraformaldehyde and subsequently permeabilized
during a 15-min incubation in 4% paraformaldehyde and 0.2% Triton
X-100 in PBS, washed, blocked and treated for 4 h with a rabbit
anti-p65 NF-B antibody (1:200) or a mouse anti-cytochrome
c antibody (1:200). The coverslips were then washed and
incubated for 1 h with a fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (1:100; Jackson ImmunoResearch) or a rhodamine (TRITC)-conjugated goat anti-mouse IgG (1:200; Jackson ImmunoResearch), respectively. Nuclei were visualized by a 5-min incubation with Hoechst
dye (5 µg/ml bisbenzimide; Roche Molecular Biochemicals). The
coverslips were mounted on microscope slides and stored protected from
light at 20 °C. The cells stained for NF-B were photographed with a Dage-MTI 100 video camera mounted on a Zeiss Axiovert microscope equipped with fluorescence optics, using a video digitizer (Snappy Video Snapshot, Play Inc., Rancho Cordova) operated through Adobe Photoshop. The cells stained for cytochrome c were observed
with a confocal microscope (Olympus IX 70), PerkinElmer Biosciences Ultraview software, spinning disk, using an Olympus ×40, 1.4 numerical aperture, oil immersion lens. Fluorescence signals were collected after
single line excitation at 543 nm (red).
80 °C. For determination of the total
glutathione content, 5 µl from the supernatants were added to 35 µl
of a buffer containing 6.3 mM EDTA in 125 mM
sodium phosphate (GAB). Then 200 µl of 0.315 mM NADPH
solution prepared in GAB (final concentration, 0.21 mM), 0.55 units of glutathione reductase, and 50 µl of 12 mM
5,5'-dithiobis-(2-nitrobenzoic acid) solution prepared in GAB (final
concentration, 2 mM) were added, and the reaction was
followed for 5 min by reading the OD at 412 nm. Concentration values
were obtained using a standard curve with known glutathione values
(0.1-100 nM) from a stock solution prepared in GAB, and
they were normalized to protein concentrations.
80 °C. On the day of the analysis, cell pellets were
solubilized in lysis buffer and centrifuged to remove debris, and equal
amounts of protein were incubated with 50 µM
N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethyl coumarin) for 1 h at 37 °C. The samples were analyzed
using an enzyme-linked immunosorbent assay plate reader with excitation of 360 ± 40 and emission of 530 ± 25.
80 °C. Protein concentrations from postnuclear
lysates and cytosols were determined using the Bio-Rad assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
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Fig. 1.
Structure of GW4869.
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Fig. 2.
Inhibition of Rat Brain N-SMase by GW4869:
Effect of varied [SM]. Delipidated rat brain N-SMase was assayed
for activity in the presence or absence of GW4869 as described under
"Experimental Procedures." A, double-reciprocal plot for
the effects of GW4869 (0-30 µM) on substrate (SM).
B, inhibition of N-SMase by GW4869 at different SM
concentrations.
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Fig. 3.
A, inhibition of Rat brain N-SMase by
GW4869: effect of varied [PS]. Delipidated rat brain N-SMase was
assayed for activity as described under "Experimental Procedures"
in the presence or absence of GW4869 at different PS concentrations.
B, inhibition of rat brain N-SMase by GW4869: effects of
varied [Mg]. Delipidated rat brain N-SMase was assayed for activity
as described under "Experimental Procedures" in the presence or
absence of GW4869 at different Mg2+ concentrations.
View larger version (19K):
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Fig. 4.
Lack of inhibition of GW4869 on
A-SMase. Cloned human A-SMase was incubated in the absence and
in the presence of increasing concentrations of GW6948 as described
under "Experimental Procedures."
Specificity of GW4869 on enzyme inhibition
Cell Studies
Effects of GW4869 on Cellular Activation of N-SMase--
To verify
the effect of GW4869 on N-SMase activity in cells, MCF7 breast cancer
cells were treated with 3 nM TNF, since in this cell line
it has been previously shown that treatment with TNF would cause
activation of N-SMase as evaluated by changes in SM and ceramide (13).
As expected, the addition of TNF caused sphingomyelin hydrolysis (Fig.
5A) and ceramide accumulation
(Fig. 5B) after 12-14 h of incubation. Importantly, the
addition of 10 µM GW4869 significantly inhibited
TNF-induced SM hydrolysis, whereas 20 µM of the compound
protected completely from the loss of SM. In the same time frame,
ceramide levels started to increase (12-14 h), and they continued to
build up during the incubation as measured by the diacylglycerol kinase
assay. The addition of GW4869 (10 and 20 µM) completely
inhibited the initial accumulation of ceramide, whereas this effect was
partially lost at later time points (24 h). These data therefore
support the inhibitory action of GW4869 on N-SMase not only in
vitro but also in a cellular model.
|
Effects of GW4869 on TNF-induced Depletion of GSH--
To localize
the site of action of GW4869 in cells, the effects of the compound on
molecular events involved in the action of TNF on N-SMase were
evaluated. One of the proposed mechanisms for the activation of N-SMase
upon TNF signaling implicates the drop in GSH levels after induction of
early caspases (1). Therefore, MCF7 cells were treated with TNF in the
presence or absence of GW4869, and GSH levels were measured (Fig.
6). As expected, TNF induced a
significant decrease of glutathione levels (60%), which occurred in
the same time frame of the previously observed SM hydrolysis (Fig.
5A). The addition of GW4869 at both 10 and 20 µM concentrations (effective in inhibiting SM hydrolysis)
did not modify glutathione levels significantly. These results suggest that the action of GW4869 occurred downstream of the drop in
glutathione.
|
Specificity of the Cellular Action of GW4869 to the N-SMase Pathway
of Ceramide Generation--
It has been recently shown that, in MCF7
cells treated with TNF, ceramide accumulation could result from both
early activation of N-SMase and later activation of the de
novo biosynthetic pathway, inhibitable by fumonisin B1 (41).
Therefore, we wondered whether the inhibitory effect of GW4869 on
ceramide accumulation (Fig. 5B) also extended to this second
ceramide-generating pathway. MCF7 cells were treated with TNF and
concomitantly pulsed with radioactive palmitate, a precursor for
ceramide synthesis. Incorporation of radioactivity into ceramide was
evaluated in the presence or absence of GW4869. As shown in Fig.
7, TNF treatment caused a 75% increase
of newly synthesized ceramide after 21 h of incubation. On the
other hand, the addition of GW4869, at either 10 or 20 µM, did not affect ceramide accumulation significantly.
These results have two important implications: first, they further
support the specificity of action of GW4869 to N-SMase-mediated
ceramide formation. Second, they show that de novo synthesis
of ceramide, which is also induced by TNF, is independent of activation
of N-SMase.
|
Lack of Effect of GW4869 on TNF-induced Activation of
NF-B--
Several molecular events triggered by TNF stimulation
have been suggested to be independent of TNF-induced accumulation of ceramide. The rapid and sustained activation of NF-B after TNF treatment and its translocation to the nucleus appears to be a major
ceramide-independent effect (42). To further verify that the effects
observed during TNF treatment in the presence of GW4869 were due to
specific interaction of the compound with the ceramide-mediated pathway, the effects of GW4869 on TNF-induced NF-B translocation were studied. MCF7 cells were treated with TNF for 30 min in the absence or presence of GW4869 (10 and 20 µM), and NF-B
activation and translocation to the nucleus were monitored by
immunocytochemical methods (Fig.
8B). As shown in the figure,
control cells exhibited a diffuse NF-B localization both in the
cytosol and in the nucleus, and the presence of the N-SMase inhibitor
at both concentrations did not affect this pattern. Thirty-minute
treatment with TNF induced a robust translocation of the cytosolic
fraction to the nucleus, and the presence of the N-SMase inhibitor did
not interfere with this phenomenon.
|
TNF-induced translocation of NF-B was also evaluated by
electrophoretic mobility shift assay (Fig. 8C). Cells were
treated with TNF for 30 min with or without GW4869, and nuclei and
nuclear proteins were extracted and processed as described under
"Experimental Procedures." As shown in the gel (left
panel), the amount of NF-B in the nuclei greatly
increased upon TNF treatment, and the presence of GW4869 did not
significantly impair it. Supershift assays (right panel) demonstrate the specificity of this translocation in
the presence or absence of GW6948 and the involvement of the p65
subunit of NF-B. Therefore, these results show that GW4869 does not
interfere with key TNF signaling effects, and they further suggest that the activation of NF-B by TNF is independent of N-SMase.
Biological Implications of N-SMase Activation in TNF- induced
Cell Death--
The above results suggest specific cellular effects of
GW4869 on activation of N-SMase but not on other key cellular effects such as activation of NF-B or depletion of GSH or even de
novo generation of ceramide. Since both the drop of GSH and the
elevation in ceramide have been correlated with TNF-induced cell death, the effects of GW4869 on cell viability were next examined. Initially, the effects of GW4869 on TNF-induced cytotoxicity were evaluated by
trypan blue exclusion assay. As shown in Fig.
9A, the presence of the
N-SMase inhibitor at 10 and 20 µM effectively reduced the number of trypan blue-positive floating cells. Similarly, GW4869 was
also able, in a dose-dependent manner, to significantly
protect from nuclear condensation after TNF treatment, as evaluated by DNA staining with Hoechst (Fig. 9B). In fact, after 20 h of treatment with TNF, 81% of the cells were positive for chromatin
condensation, whereas the presence of 10 and 20 µM GW4869
during the incubation reduced this percentage to 57.2 and 34%,
respectively.
|
To further verify the action of GW4869 on N-SMase, we investigated
whether exogenous ceramide can bypass the effects of the inhibitor.
Therefore, MCF7 cells were treated with either TNF or C6-ceramide, in
the presence or absence of GW4869, and cell viability was evaluated
using the MTT assay. As shown in Fig. 10A, TNF treatment
significantly affected cell viability, reducing it by 35% after
48 h. The addition of GW4869 efficiently protected cell viability
at both 10 and 20 µM. MCF7 cells were then treated with
C6-ceramide. In response to treatment with C6-ceramide, cell viability
was significantly impaired (34%), but in this case the presence of
GW4869 was unable to prevent this effect (Fig. 10B). Therefore, these results show that the GW4869 does not interfere with
molecular events leading to cell death downstream of ceramide formation, further supporting its action at the N-SMase level.
|
To gain more insight on the specific components of the apoptotic
pathway regulated by GW6948, cell morphology was evaluated by EM
studies conducted in cells treated with TNF in the absence and in the
presence of the N-SMase inhibitor (Fig.
11). As shown in the figure, cells
treated with TNF (Fig. 11, E and F) showed massive chromatin condensation compared with control cells
(A and B), whereas the presence of the inhibitor
significantly reduced this condensation (G and
H). It is interesting to note that in this latter condition,
the nuclear changes were variable among the cellular population. In
fact, some cells showed no signs of chromatin condensation
(G), whereas some others showed partially condensed
chromatin only at peripheral areas of the nuclei (H). From
these studies, other morphological alterations in TNF-treated cells
could be also clearly appreciated, such as swollen mitochondria (E and F), vacuolization of the cytoplasm
(E and F), extensive blebbing of the plasma
membrane (F), and disruption of cellular junctions
(E). In the presence of the N-SMase inhibitor, these changes
were markedly reduced, in particular the blebbing of the plasma
membrane (G and H). It was important to note,
however, that the inhibitor was not able to prevent the formation of
the large molecular weight fragments of condensed chromatin peripheral to the nuclei (H).
|
In order to verify that these morphological changes were part of an
ongoing apoptotic process, we evaluated the effect of TNF treatment on
activation of late/effector caspases, thought to be responsible for
nuclear fragmentation. As shown in Fig. 12 and in agreement with previous
reports (13, 30, 43, 44), treatment of MCF7 cells with TNF caused
cleavage of PARP, in MCF7 a substrate for active caspase 7, with the
formation of the characteristic 85-kDa fragment. The presence of the
N-SMase inhibitor prevented, in a dose-dependent manner,
the TNF-induced PARP cleavage. In fact, after 12 h of treatment,
preincubation with 10 µM GW4869 was able to partially
block the appearance of the 85-kDa fragment, whereas 20 µM of the inhibitor completely prevented the cleavage. These results clearly suggest a role for ceramide generated through N-SMase in activating downstream caspases.
|
To further investigate the effects of GW4869 late/effector caspases, we evaluated the effect of pretreatment with the N-SMase inhibitor on caspase 6 processing. Therefore, MCF7 cells were treated with TNF in the presence or absence of 20 µM GW4869. After 14 h, cells were collected, and Western blotting was performed on cytosolic proteins using antibodies recognizing the full-length caspase 6. As shown in Fig. 12, TNF induced processing of caspase 6, whereas the addition of the N-SMase inhibitor effectively blocked it. These results suggest that inhibition of N-SMase activation prevents activation of late caspases. Next, in vitro activity of DEVDase caspases was assayed after treating cells with TNF in the presence or absence of the N-SMase inhibitor in order to evaluate a more generalized activation of effector caspases. As shown in Fig. 12, the presence of increasing concentrations of GW4869 (10 and 20 µM) resulted in almost complete inhibition of activation of DEVDase caspases induced by TNF, confirming and extending the results obtained from the Western blots.
One of the receptor-mediated mechanisms for the activation of late
caspases involves mitochondrial dysfunction, characterized by release
of cytochrome c and subsequent activation of caspase 9 (45-48). Thus, we wondered if N-SMase activity was involved in mediating these events. Therefore, MCF7 cells were treated with TNF in
the presence or absence of GW4869, and its effects on cytochrome c release were evaluated by immunocytochemistry and confocal
microscopy. As shown in Fig.
13A, untreated cells
exhibited a punctate pattern characteristic of the mitochondrial
network, whereas treatment with TNF induced the appearance of a diffuse
staining characteristic of cytosolic localization. Importantly, the
presence of the N-SMase inhibitor significantly and in a
dose-dependent manner protected the release of cytochrome
c from mitochondria into the cytosol; 10 and 20 µM of GW4869 reduced by 46 and 62% the number of
positive cells, respectively. In support of the effects on cytochrome
c release, the presence of the N-SMase inhibitor also
protected from activation of caspase 9 (Fig. 13B). Treatment
with TNF resulted in the appearance of the characteristic proapoptotic
fragment and the presence of the N-SMase inhibitor efficiently
prevented processing of caspase 9.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Here we report the identification of GW4869 as a novel inhibitor of the Mg2+-dependent N-SMase and its characterization and biological effect in a cellular system of cytokine-induced apoptosis. Importantly, evidence is provided of specificity of action of GW4869 on N-SMase activity both in vitro and in vivo. Finally, the use of this novel N-SMase inhibitor provided strong evidence implicating activation of N-SMase in the regulation of a number of biological processes induced by TNF.
During a high throughput screen with a preparation of delipidated rat brain N-SMase, we successfully identified GW4869, a molecule that exhibited significant and specific inhibitory activity. The compound is a noncompetitive inhibitor of the enzyme with an IC50 of 1 µM. GW4869 showed no or minor inhibitory activity versus other hydrolytic enzymes, such as bacterial phosphatidylcholine-PLC and bovine protein phosphatase 2A, and it showed significantly higher activity versus the rat brain enzyme compared with the human lyso-PAF PLC (Table I). Importantly, GW6948 showed no inhibition of the human acid SMase.
The inhibitory activity of GW4869 against N-SMase observed in vitro (Figs. 2-4 and Table I) was also confirmed at a cellular level. GW4869 prevented SM hydrolysis induced by TNF treatment and partially inhibited ceramide accumulation (Fig. 5). These effects have been observed using concentrations ranging from 5 to 20 µM. The requirement for higher concentrations for cellular action could be due to limitation in permeability through the plasma membrane.
Specificity of action of GW4869 to the N-SMase was also confirmed
in vivo by multiple lines of evidence. 1) GW4869 only
targeted ceramide formation from the N-SMase pathway. In MCF7 cells,
TNF treatment activates at least two major routes for ceramide
generation, the N-SMase and the de novo biosynthetic
pathways (38). GW4869 completely inhibited ceramide accumulation only
at an early phase of incubation (12-18 h), when at the same time it
also inhibited SM hydrolysis (Fig. 5). At a later time, corresponding
to the activation of the de novo pathway, the inhibitor no
longer prevented the rise of ceramide levels. When the effects of the
inhibitor were directly tested on this later ceramide-generating
pathway, no effects were observed on de novo incorporation
of palmitate into ceramide (Fig. 7). Since the de novo
pathway requires many enzymes including serine palmitoyl transferase
and ceramide synthase, these results demonstrate that GW6948 does not
affect these enzymes. It is also interesting to note that, in addition
to demonstrating specificity of action of GW4869 on N-SMase-generated
ceramide, these results also indicate that the two pathways of ceramide generation are independent. 2) GW4869 did not inhibit the drop in
glutathione induced by TNF treatment, which has been recently shown to
precede the activation of N-SMase in response to TNF (13, 49-51).
These results also add to the cellular specificity of action of GW4869
(Fig. 6). 3) GW4869 did not inhibit ceramide-mediated cell death. This
indicates that indeed the target of GW4869 is localized upstream of
ceramide generation and that the N-SMase inhibitor does not affect
directly any of the targets downstream of ceramide action (such as
caspases, cytochrome c, nucleases, etc.). 4) GW4869 did not
affect other molecular events triggered by TNF that are known to be
independent of ceramide accumulation. Thus, the lack of effects on
NF-B demonstrates that GW4869 does not interfere with initial events
in TNF action (including interaction with the TNF receptors, activation
of Nik, Ikk, or the proteosome). Additionally, GW4869 did not affect
TNF-induced prostaglandin production in both A549 lung cancer cells and
L929 murine fibroblasts, thus excluding a possible effect of the
compound on either cPLA2 or
cyclooxygenase.2
The use of GW4869 as a specific inhibitor generated specific insight into the molecular mechanisms that are dependent on activation of N-SMase. Thus, inhibition of N-SMase not only blocked PARP cleavage (target for caspase 7) but also prevented the activation of other effector caspases such as caspase 6 and caspase 9. Processing of procaspase 9 occurs by autocleavage as the result of the formation of the so-called "apoptosome," a complex constituted by cytochrome c released from mitochondria, apoptotic protease-activating factor-1, and procaspase 9 (52, 53). Therefore, the possibility that activation of N-SMase leads to the induction of effector caspases by targeting specific mitochondrial events was verified and confirmed, since inhibition of N-SMase partially, but significantly, prevented the release of cytochrome c from the mitochondria (Fig. 13). The observation that the activation of N-SMase promotes cell death through mitochondrial dysfunction is in agreement with results obtained in MCF7 overexpressing the Bcl2 protein. Indeed, the overexpression of Bcl2 has been shown to almost completely prevent TNF-induced cell death (27) and PARP proteolysis (13), only moderately blocking ceramide formation (27), and without inhibition of either the drop of glutathione or SM hydrolysis (13). In addition, these results are in agreement with some studies that previously reported the ability of short chain ceramide analogs to induce cytochrome c release in both isolated mitochondria (54) and intact cells (55, 56).
Although these results start to shed light on the possible biological targets of ceramide generated through the N-SMase pathway, they also raise a number of important questions that require further evaluation. First, how does the ceramide from the N-SMase signal to mitochondria? It has been recently demonstrated that targeting of an N-SMase to mitochondria induced accumulation of ceramide that was sufficient to induce mitochondrial dysfunction and signal cell death (57). Therefore, one possible scenario implicates the activation by TNF of an N-SMase isoform, which resides in mitochondria or in close proximity. Second, what are the direct effectors of the mitochondrial dysfunction induced by accumulation of ceramide? Recent studies suggest a few candidates. For example, it has been shown that ceramide induces conformational changes of the Bax protein and synergizes with Bax, altering mitochondrial functions (58). On the other hand, ceramide was found to specifically activate a mitochondrial protein phosphatase 2A, which rapidly and completely dephosphorylated Bcl-2, leading to cell death (59).
It is also important to note that inhibition of N-SMase did not completely prevent either the formation of ceramide (Fig. 5B) or some of the biological effects induced by TNF in MCF7, such as cytochrome c release (Fig. 13A) and nuclear condensation (Fig. 9B). Interestingly, the predominant peripheral condensation of the chromatin observed in MCF7 treated with TNF in the presence of the N-SMase inhibitor (Fig. 11H) closely resembles the pattern of nuclear changes induced by activation of the apoptosis-inducing factor-mediated pathway (60). These results suggest that this specific process, which is known to be independent of caspases, may also be independent of N-SMase.
In conclusion, by using a newly characterized inhibitor of N-SMase, we
show for the first time that activation of the N-SMase leads to
mitochondrial dysfunction and that it is required for the full
development of the cytotoxic program induced by TNF.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Charles Chalfant and Patrick Roddy for technical assistance with the protein phosphatase assay.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grants GM 43825 and CA 87584 (to Y. A. H.) and AG 16583 (to L. M. O.).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.
¶ Present address: Dept. of General Pathology, Universita' degli Studi di Milano, 20133 Milano, Italy.
Recipient of the Merck Company Foundation and Banyu Fellowship
Awards in Lipid Metabolism and Atherosclerosis.
** Present address: Dept. of Medicine, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573, Japan.
¶¶ To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, 173 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322; E-mail: hannun@musc.edu.
Published, JBC Papers in Press, August 1, 2002, DOI 10.1074/jbc.M206747200
2 B. Pettus and Y. A. Hannun, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
TNF, tumor necrosis
factor 
;
SMase, sphingomyelinase;
N-SMase, neutral sphingomyelinase;
A-SMase, acid sphigomyelinase;
PARP, poly(ADP-ribose) polymerase;
PS, phosphatidylserine;
SM, sphingomyelin;
PAF, platelet-activating factor;
PLC, phospholipase C;
FBS, fetal bovine serum;
MSA, methane sulfonic
acid;
PBS, phosphate-buffered saline;
TRITC, tetramethylrhodamine
isothiocyanate;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Hannun, Y., and Luberto, C. (2000) Trends Cell Biol. 10, 73-80[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Levade, T., and Jaffrezou, J. P. (1999) Biochim. Biophys. Acta 1438, 1-17[Medline] [Order article via Infotrieve] |
| 3. | Mathias, S., Pena, L. A., and Kolesnick, R. N. (1998) Biochem. J. 335, 465-480 |
| 4. | Levade, T., Andrieu-Abadie, N., Segui, B., Auge, N., Chatelut, M., Jaffrezou, J. P., and Salvayre, R. (1999) Chem. Phys. Lipids 102, 167-178[CrossRef][Medline] [Order article via Infotrieve] |
| 5. |
Zhang, Y.,
Mattjus, P.,
Schmid, P. C.,
Dong, Z.,
Zhong, S., Ma, W. Y.,
Brown, R. E.,
Bode, A. M.,
and Schmid, H. H.
(2001)
J. Biol. Chem.
276,
11775-11782 |
| 6. | Bilderback, T. R., Gazula, V. R., and Dobrowsky, R. T. (2001) J. Neurochem. 76, 1540-1551[CrossRef][Medline] [Order article via Infotrieve] |
| 7. |
Boucher, L. M.,
Wiegmann, K.,
Futterer, A.,
Pfeffer, K.,
Machleidt, T.,
Schutze, S.,
Mak, T. W.,
and Kronke, M.
(1995)
J. Exp. Med.
181,
2059-2068 |
| 8. |
Kirschnek, S.,
Paris, F.,
Weller, M.,
Grassme, H.,
Ferlinz, K.,
Riehle, A.,
Fuks, Z.,
Kolesnick, R.,
and Gulbins, E.
(2000)
J. Biol. Chem.
275,
27316-27323 |
| 9. |
