Received for publication, August 13, 2002, and in revised form, November 21, 2002
Apoptosis is a highly regulated process
that plays a critical role in neuronal development as well as the
homeostasis of the adult nervous system. Vanadate, an environmental
toxicant, causes developmental defects in the central nervous
system. Here, we demonstrated that vanadate induced apoptosis in
cultured cerebellar granule progenitors (CGPs). Treatment of cultured
CGPs with vanadate activated ERKs and JNKs but not p38 MAPK and also
induced c-Jun phosphorylation. In addition, vanadate induced FasL
production, Fas (CD95) aggregation, and its association with the
Fas-associated death domain (FADD), as well as the activation of
caspase-8. Furthermore, vanadate generated reactive oxygen species
(ROS) in CGPs; however, ROS was not involved in vanadate-mediated MAPK
activation. Vanadate-induced FasL expression was
ROS-dependent but JNK-independent. In contrast, vanadate-elicited Fas aggregation and Fas-FADD association, as well as
caspase-8 activation, were dependent on JNK activation but were
minimally regulated by ROS generation. The hydrogen peroxide scavenger,
catalase, blocked vanadate-induced FasL expression and partially
mitigated vanadate-induced cell death. On the other hand, dominant
negative FADD and caspase-8 inhibitor completely eliminated
vanadate-induced apoptosis. Thus, JNK signaling plays a major role in
vanadate-mediated activation of the Fas-FADD-caspase-8 pathway that
accounts for vanadate-induced apoptosis of CGPs.
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INTRODUCTION |
Apoptosis, or programmed cell death, plays an important role in
shaping the developing nervous system, removing unwanted neurons, and
establishing correct synaptic connections with the targets they
innervate (1-4). In the adult, inappropriate apoptosis in the central
nervous system may contribute to a number of neurodegenerative diseases
including Alzheimer's disease, Parkinson's disease, and stroke
(5-8). Understanding the mechanisms that govern neuronal apoptosis
could lead to more effective therapies for these disorders.
Mitogen-activated protein kinases
(MAPKs)1 play an instrumental
role in the transmission of signals from cell surface receptors and
environmental cues to the transcriptional machinery. Three major MAPKs
have been identified: the extracellular signal-regulated kinases
(ERKs), the c-Jun NH2-terminal protein kinases (JNKs), and
the p38 mitogen-activated protein kinase (p38 MAPK). ERKs are mainly
activated by growth factors and are involved in the regulation of cell
proliferation (9, 10). Unlike ERKs, JNKs and p38 MAPK are most potently
activated by environmental stresses. JNKs are identified through their
ability to phosphorylate the N-terminal stimulatory sites of c-Jun
(11). Along with JNKs, p38 MAPKs are implicated in the regulation of
cell death. JNKs and p38 MAPK may regulate apoptosis positively or
negatively depending on cell type (12-15). However, the mechanisms by
which JNKs and p38 MAPKs regulate neuronal apoptosis are not clear.
Conflicting results have been published regarding the role of JNKs and
p38 MAPK signaling in the control of neuronal apoptosis (16-26).
The trace metal vanadium is widely distributed in the environment (27,
28). It exists in oxidation states ranging from
1 to +5. Among these
oxidation states the pentavalent state is the most stable form. In
mammalian systems vanadium, the vanadate, is the most toxic form.
Occupational exposure to vanadium is common in oil-fired electrical
generating plants and petrochemical, steel, and mining industries (28,
29). Vanadate-containing compounds exert toxic effects on a wide
variety of biological systems (30-34). Developmental exposure to
vanadium is teratogenic and results in profound defects in the central
nervous system (35-40). The objective of this study was to determine
the mechanisms of vanadate-induced damages to the developing central
nervous system and define the role of MAPKs in the regulation of
neuronal apoptosis.
The developing cerebellum is one of the regions that is most
susceptible to environmental insults, and vanadium compounds are
deposited in the human cerebellum (41). Cerebellum granule neurons, the
major cerebellar neuronal population, are generated in the
proliferative external germinal layer of the cerebellum during the
first 2-3 postnatal weeks in the rat (42, 43). During the early
postnatal period, cells in the external germinal layer undergo
extensive proliferation to generate a large pool of cerebellar granule
progenitors (CGPs). The developing CGPs then exit the cell cycle and
migrate inward past Purkinje cell bodies to their final destination in
the internal granule layer, where they differentiate into cerebellar
granule neurons (42, 43). The CGPs isolated from 3-5-day (a time when
CGPs are being generated and proliferating in vivo)-old rat
pups retain their proliferative capability in vitro and are
able to differentiate into neurons (44-47). This in vitro
system offers an excellent model for study of neuronal development.
Here, we demonstrate that vanadate induces apoptosis of CGPs. JNK
signaling and the death receptor Fas (CD95) play a central role in
vanadate-mediated neuronal death.
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EXPERIMENTAL PROCEDURES |
Reagents
Sodium metavanadate (vanadium (V), NaVO3, or
vanadate, catalog no. 2839-1) was purchased from Sigma.
Caspase-3 inhibitor Ac-DMQD-CHO, caspase-8 inhibitor Z-IETD-FMK, and
caspase-9 inhibitor ZlEHD-FMK were obtained from Calbiochem. Anti-Fas
antibodies, SM1/23 and FL-335, were purchased from Alexis Biochemicals
(San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz, CA),
respectively. Fas-Fc fusion protein was purchased from R&D Systems
(Minneapolis, MN). Anti-Fas ligand (FasL) (N-20) and anti-FADD
antibodies (H-181) were obtained from Santa Cruz Biotechnology. Human
recombinant FasL was obtained from Upstate Biotechnology Inc. (Lake
Placid, NY). Deferoxamine, superoxide dismutases, and sodium formate
were purchased from Sigma. Catalase was obtained from Roche Molecular Biochemicals. The inhibitors for protein tyrosine phosphatase (
-bromo-4-hydroxyacetophenone), p38 MAPK (SB202190), and MEK1 (PD98059) were purchased from Calbiochem, and a selective JNK inhibitor
(D-JNKI1) was purchased from Alexis Biochemicals.
Culture of CGPs
The CGPs were isolated from the cerebella of 3-day-old
Sprague-Dawley rats (Hilltop Laboratory Inc., Scottsdale, PA) using a
previously described procedure (47, 48). The purity of CGPs generated
using this procedure is greater than 95.0%. Isolated CGPs were plated
into poly-D-lysine (50.0 µg, Sigma)-coated culture wells
or dishes at a density of 3.2 × 104/cm2.
The cultures were grown in Eagle's minimal essential medium containing
the following supplements: 10.0% fetal bovine serum, 25.0 mM KCl, 1.0 mM glutamine, 33.0 mM
glucose, and penicillin (100 units/ml)/streptomycin (100.0 µg/ml).
Cells were incubated at 37 °C in a humidified environment containing
5.0% CO2.
Cell Growth and Proliferation
The number of viable cells in culture was determined by the MTT
assay (catalog no. 1 465 007, Roche Molecular Biochemicals). Briefly,
the CGPs were plated into 96-well microtiter plates at a density of
3.2 × 104/cm2. 10 µl of MTT labeling
reagent was added to each well, and plates were incubated at 37 °C
for 4 h. After MTT incubation the cultures were solubilized, and
spectrophotometric absorbance of the samples was detected by a
microtiter plate reader. The wavelength to measure the absorbance of
formazan product was 570 nm with a reference wavelength of 750 nm. We
have demonstrated that the MTT assay is an accurate method to determine
the number of viable cells (47, 49). Cell proliferation was determined
by pulse labeling of 5-bromo-2'-deoxyuridine (BrdUrd) (47, 50).
Briefly, cells were plated into wells of removable chamber slides at
the density of 3.2 × 104/cm2 and exposed
to 20.0 µM BrdUrd (Sigma) for 12 h. BrdUrd positive cells were visualized immunohistochemically using an anti-BrdUrd monoclonal antibody (Sigma). The numbers of BrdUrd positive and negative cells within an area of 0.5 × 0.5 mm were counted. Five fields were chosen at random in each slide and counted. A BrdUrd labeling index (the number of labeled cells divided by the total number
of cells counted) was calculated.
Apoptosis Assays
Cellular DNA Fragmentation ELISA--
This assay is based on
quantification of accumulation of DNA fragments in the cytoplasm of
apoptotic cells. Accumulation of cytosolic histone-bound DNA fragments
was quantified using a commercial ELISA kit (catalog no. 1 544 675, Roche Molecular Biochemicals). The measurement of apoptosis, which was
demonstrated previously, was sensitive, and the outcome was
consistent with morphometric indices of apoptosis or the terminal
deoxynucleotidyl transferase-mediated dUTP-biotin nick
end-labeling assay (47, 51).
Nuclear Morphology--
Cell death was also determined by
counting the amount of cells containing condensed or fragmented nuclei
in a blinded manner (52). Briefly, cells were fixed with 2.0%
paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room
temperature. Cell membranes were permeabilized by treating the cells
with 0.10% Triton X-100 in PBS for 15 min and then stained with a DNA
dye, TOTO-3 iodide (3.0 µg/ml; Molecular Probes Inc., Eugene, OR), for 30 min at 37 °C to visualize nuclear morphology. After being washed with PBS, the cells were examined under a Zeiss LSM 510 confocal
microscope. Apoptotic neurons were determined by counting the cells
containing condensed or fragmented nuclei in 4-5 fields/well. At least
200 cells were counted.
Caspase Activity
Caspase activity was determined using commercial fluorometric
protease assay kits. Kits for assaying caspase-3 and caspase-8 activation were obtained from Santa Cruz Biotechnology and the caspase-9 activity kit from R&D Systems. The activation of caspases was
determined for their protease activity by the addition of a
caspase-specific substrate peptide that is conjugated to the fluorescent reporter molecule 7-amino-4-trifluoromethyl coumarin. The
cleavage of the substrate peptide by specific caspase releases the
fluorochrome that emits fluorescence at 505 nm when excited by light
with a 400-nm wavelength. The level of caspase enzymatic activity is
directly proportional to the fluorescence signal detected with a fluorometer.
Immunoblot and Immunoprecipitation Analysis
Cell lysates (40-100 µg of protein) were resolved by SDS-PAGE
on 8-12% polyacrylamide gels, and separated proteins were transferred to nitrocellulose membranes. After blocking with 5.0% nonfat milk in
TPBS (0.010 M PBS, pH 7.4, and 0.10% Tween 20) at
room temperature for 1 h, the membranes were probed with various
antibodies directed against activated MAPKs or phosphorylated tyrosine.
These antibodies include anti-phospho-c-Jun, anti-phospho-p38
MAPK, anti-phospho ERK, anti-JNK1, anti-c-Jun, anti-p38, and anti-ERK,
which were obtained from Santa Cruz Biotechnology. Anti-phospho-JNK
antibody was purchased from Promega (Madison, WI) and
anti-phosphotyrosine antibody from Cell Signaling Technology, Inc.
(Beverly, MA). The antibody-antigen complexes were visualized by
enhanced chemiluminescence (Amersham Biosciences). The
blots were stripped and reprobed with an anti-
-actin antibody (Santa
Cruz Biotechnology). The variation in loading was normalized against
the amount of actin expression in each lane. Each experiment was
repeated three to four times for each primary antibody. The relative
amount of specific protein imaged on the films was quantified using a
Sigma Gel program (Jandel, San Rafael, CA). Fas was
immunoprecipitated from cell lysates (200.0 µg of total protein) by
an anti-Fas antibody (Santa Cruz Biotechnology) using a previously
described method (53). Immunoprecipitates were electrophoretically
separated and transferred to nitrocellulose membranes. FADD associated
with Fas was examined by immunoblotting using an anti-FADD antibody.
Fas Aggregation
Fas aggregation was determined using a previously described
method (54, 55). Basically, when antibody is limiting (0.1-0.5 µg/ml), more molecules of the Fas will be immunoprecipitated if aggregation occurs after vanadate treatment. In contrast, when antibody
is not limiting (5-10 µg/ml), equal amounts of Fas should be
immunoprecipitated. Briefly, cells were treated with vanadate (25.0 µM, 6 h), resuspended in 500 µl of PBS, and then
incubated with 2.0 mM cross-linker
3,3'-dithiobis-[sulfosuccinimidyl propionate] (Sigma) for 15 min on
ice. The reaction was quenched with 10.0 mM ammonium
acetate for 10 min and the cells were washed with PBS and then lysed.
Fas was immunoprecipitated using either limiting or excess amounts of
anti-Fas antibody and detected by immunoblot.
Cell Transfection
The pcDNA3.0/DN-FADD vector (kindly provided by Dr. V. M. Dixit, Genetech, San Francisco, CA) carries a truncated FADD
cDNA lacking the death effector domain (DED) region (56,
57). pEGFP-C3 encoding the green fluorescent protein was obtained from
Clontech Laboratories (Palo Alto, CA). The cells
were transfected with pEGFP-C3 (0.30 µg) and DN-FADD (1.0 µg) or an
empty pcDNA3.0 vector (1.0 µg) using LipofectAMINE 2000 reagent
(Invitrogen) according to the manufacturer's instructions. One day
after the transfection the CGPs were treated with vanadate (25.0 µM) for 24 h. Nuclei were stained with TOTO-3
iodide, and apoptotic neurons in total transfected (green fluorescent
protein-positive) cells were determined as described above.
Electron Spin Resonance (ESR) Measurements
The ESR spin trapping technique with 5,5-dimethyl-1-pyrroline
N-oxide (DMPO) as the spin trap was used to detect free
radical generation. This technique involves the formation of an adduct of a short-lived radical with a diamagnetic compound (spin trap) to
generate a relatively long-lived free radical product (spin adduct),
which can be studied by conventional ESR. The intensity of the spin
adduct signal corresponds to the amount of short-lived radicals
trapped, whereas the hyperfine couplings of the spin adduct
are characteristics of trapped radicals. The spectra were recorded
using a Varian E9 ESR spectrometer and a flat cell assembly as
described previously (58). Hyperfine couplings were measured (to 0.1 G)
directly from magnetic field separation using potassium tetraperoxochromate (K3CrO8) and
1,1-diphenyl-2-picrylhydrazyl as reference standards. 500.0 µl of
cell suspension (1 × 106/0.5 ml) was mixed with 500.0 µl of PBS containing 100.0 mM DMPO and 1.0 mM
vanadate with/without ROS scavengers. The reaction mixture (500.0 µl)
was transferred to a flat cell for ESR measurement.
Statistical Analysis
Treatment groups were compared using analysis of variance.
In cases where significant differences (p < 0.05)
were detected, specific post hoc comparisons between treatment
groups were examined with the Student-Newman-Keuls tests.
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RESULTS |
Vanadate-induced Apoptosis in Cultured CGPs--
Vanadate exposure
reduced the number of viable cells in a
concentration-dependent manner (Fig.
1A). Concomitantly, vanadate increased apoptotic death, which was determined by a cellular DNA
fragmentation ELISA in a dose-dependent manner (Fig.
1B). At a concentration of 25.0 µM, vanadate
treatment (24 h) increased death rate by 3.4-fold. Vanadate-induced
apoptosis was verified with an analysis of nuclear morphology by TOTO-3
iodide staining; it showed a 3.1-fold increase in apoptotic rate after
vanadate treatment (Fig. 1, C and D). Time course
analysis revealed that maximal apoptosis occurred 24-48 h after
vanadate treatment (Fig. 1E). Because CGPs proliferate in
culture, we sought to determine whether vanadate affects the
proliferation of CGPs. As shown in Fig. 1F, vanadate (25.0 µM) produced a small but statistically significant
decrease (
19%; p < 0.05) of BrdUrd positive cells. In contrast, at this concentration vanadate decreased the number of
CGPs by more than 70%. When these facts are taken together, it
can be concluded that vanadate-induced depletion of cell number was
mainly caused by the cell death.

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Fig. 1.
Effects of vanadate on the survival and
proliferation of CGPs. The CGPs were plated at a density of
3.2 × 104/cm2. At 24 h after plating
cells were treated with vanadate. A, effect of vanadate on
cell number. Cells were exposed to vanadate (0-100.0 µM)
for 48 h, and the number of viable cells was determined with the
MTT assay. B, vanadate-induced apoptosis. Cells were exposed
to vanadate (0-100.0 µM) for 24 h, and apoptosis
was determined by a DNA fragmentation ELISA. C, assaying
apoptosis by nuclear morphology. Cells were exposed to vanadate (25.0 µM) for 24 h and stained with the DNA dye TOTO-3
iodide (3.0 µg) for 30 min. Cells were examined under a confocal
microscope. D, relative apoptosis shown in C was
quantified by determining the nuclei fragmentation and condensation as
described under "Experimental Procedures." E, time
course of vanadate-mediated apoptosis. Cells were exposed to vanadate
(25.0 µM) for various durations (6-72 h), and apoptosis
was determined with a DNA fragmentation ELISA. F, DNA
synthesis. Cells were treated with vanadate (25.0 µM) for
24 h. Subsequently, cells were exposed to BrdUrd (20.0 µM) for 12 h. The incorporation of BrdUrd was
determined immunohistochemically as described under "Experimental
Procedures." Each data point (bars; ± S.E.) is the mean
of 3-4 independent trials. *, denotes a statistically significant
difference between control and treated groups (p < 0.05).
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Vanadate-mediated MAPK Activation--
Vanadate produced a
sustained activation of JNK1 but did not affect JNK1 expression (Fig.
2A). The antibody applied
reacts with both JNK1 and JNK2 (Promega, catalog no. V7932), but only phosporylated JNK1 was detected. The activation lasted for at least
12 h. As a result of JNK activation, vanadate induced
phosphorylation of c-Jun, a main target of JNKs, in a pattern similar
to that of JNK1 (Fig. 2A). Vanadate had little effect on
c-Jun expression. Vanadate also produced a transient activation of
ERK1/2 (Fig. 2B). The activation of ERK1/2 was evident 15 min after vanadate treatment, and the effect diminished after 2 h.
Vanadate had little effect on the expression of ERK1/2. We also
examined the effect of vanadate on p38 MAPK. Vanadate did not
significantly alter either the expression or the activation of p38 MAPK
in CGPs (data not shown). Vanadium compounds may serve as protein
tyrosine phosphatase (PTP) inhibitor and subsequently enhance protein
tyrosine phosphorylation (59, 60). We then examined the effect of
vanadate on protein tyrosine phosphorylation of total cellular
extracts. As shown in Fig. 2, C and D, vanadate
treatment increased tyrosine phosphorylation on a number of proteins
including JNK1 as determined by immunoprecipitation.

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Fig. 2.
Vanadate-mediated activation of MAPK and
protein tyrosine phosphorylation. The CGPs were grown in a
serum-free medium for 24 h and treated with vanadate (25.0 µM) for various lengths of time. The activation of MAPKs
and protein tyrosine phosphorylation was determined with immunoblotting
using specific antibodies. After detection of phosphorylated forms of
MAPKs, the blots were stripped and reprobed with antibodies directed
against nonphosphorylated kinases. A, JNK and c-Jun
activation; B, ERK activation; C, protein
tyrosine phosphorylation; D, tyrosine phosphorylation on
JNK1. Cells were exposed to vanadate with/without catalase for 6 h, and JNK1 was immunoprecipitated using a specific antibody. The
immunoprecipitates were assayed for tyrosine phosphorylation
(top panel) or JNK1 expression (bottom
panel). The experiments were replicated three times.
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Vanadate-induced FasL Production, Fas-FADD Association, Fas
Aggregation, and Caspase Activation--
Because vanadate induced
apoptotic death of CGPs (Fig. 1), we sought to determine the mechanisms
of vanadate-induced damage. Death receptor Fas is an apoptotic sensor
that transmits a death signal (61, 62). The adapter molecule
FADD couples with the death receptor Fas to procaspase-8 (63). As shown
in Fig. 3A, vanadate produced
a concentration-dependent up-regulation in the expression
of FasL. Although vanadate did not alter the expression of Fas and
FADD, it induced an association between these proteins (Fig.
3B). Vanadate-induced activation of Fas was further
confirmed by the evidence showing an increase in Fas aggregation (Fig.
3C). As shown in Fig. 3C, vanadate induced a
JNK-dependent Fas aggregation. Because caspases play a
critical role in the initiation of apoptosis, we sought to determine
whether vanadate activates caspases. As shown in Fig.
4, vanadate treatment significantly
(p < 0.05) activated caspases-3 and -8, but had little
effect on caspase-9 (Fig. 4).

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Fig. 3.
Effects of vanadate on FasL, Fas, and
FADD. A, expression of FasL, Fas, and FADD. The CGPs
were treated with vanadate (0-40.0 µM) for 24 h,
and total proteins were isolated. Equal amounts of proteins (60.0 µg)
were subjected to immunoblot analysis. After the detection of FasL,
Fas, and FADD, the blots were stripped and reprobed with an anti-actin
antibody. B, vanadate-mediated association between Fas and
FADD. Cells were treated with vanadate (25.0 µM) for
6 h, and proteins were isolated. Cell lysates were
immunoprecipitated with an anti-Fas antibody and probed with an
anti-FADD antibody. C, vanadate-mediated Fas aggregation.
Cells were treated with vanadate (25.0 µM) for 6 h
in the presence/absence of D-JNKI1 or catalase. Cell
lysates were treated with a cross-linking reagent and
immunoprecipitated using an anti-Fas antibody under antibody-limiting
conditions (top panel) or antibody-excess conditions
(bottom panel). Panels on the right were the
microdensitometrical quantification of relative expression. Each data
point (bars; ± S.E.) is the mean of three independent
trials. *, denotes a statistically significant difference
between control and treated groups (p < 0.05).
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Fig. 4.
Effects of vanadate on caspase activity.
The CGPs were treated with vanadate (25.0 µM) for 24 h. The activity of caspase-3, -8, and -9 was determined as described
under "Experimental Procedures." Each data point (bars; ± S.E.) is the mean of four independent trials. *, denotes a
statistically significant difference between control and treated groups
(p < 0.05).
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Vanadate-induced ROS Generation--
ROS is involved in
apoptosis (64). Vanadate is able to generate a whole spectrum of
ROS, i.e. O
, H2O2 and
·OH (65). To determine whether vanadate-induced apoptosis of CGPs is mediated by ROS, we first examined the ROS generation in the
vanadate-treated cells. The ability of vanadate to generate ·OH
radicals was examined using an ERS spin trapping method with DMPO as
the spin trap. As shown in Fig. 5,
vanadate treatment generated a typical ESR spectrum of free radicals.
The spectrum consists of a 1:2:2:1 quartet with splittings of
aH = aN = 14.9 G, where aN and
aH denote hyperfine splitting of the nitroxyl nitrogen and
-hydrogen, respectively. On the basis of these splittings and the
1:2:2:1 line shape, this spectrum was assigned to the DMPO/·OH
adduct, which is evidence for ·OH radical generation. The
addition of catalase, a specific H2O2 scavenger, completely eliminated the ·OH radical, indicating
that H2O2 was generated in vanadate-stimulated cells and was a precursor of ·OH generation. Sodium formate, a
scavenger of ·OH radical, and deferoxamine, a metal chelator,
decreased the intensity of the radical signal. In contrast,
superoxide dismutase, an O
scavenger the function of which is
to convert O
to H2O2, increased the
DMPO/·OH adduct signal. The results were similar in three
independent experiments. The finding indicated that vanadate was
able to generate a whole spectrum of ROS in CGPs.

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Fig. 5.
Measurement of vanadate-induced ROS
generation by ESR. The CGPs (1 × 106) were
incubated in PBS containing 100.0 mM DMPO and 1.0 mM vanadate with or without different ROS scavengers as
indicated. ESR spectra were recorded for 30 min. The final
concentrations for ROS scavengers were: catalase, 2000 units/ml;
superoxide dismutase, 5.0 µg/ml; formate, 50.0 mM; and
deferoxamine, 2.0 mM.
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Vanadate-induced FasL Production and Fas-FADD Association Involve
Different Mechanisms--
Catalase, a specific
H2O2 scavenger, eliminated vanadate-induced ROS
generation (Fig. 5); however, it did not affect vanadate-mediated JNK
and ERK activation (Figs. 2D and
6). D-JNKI1, a specific inhibitor for
JNK1/2 (66), and PD98059, an inhibitor for MEK1, completely blocked
vanadate-induced phosphorylation of c-Jun and ERK1/2, respectively.
Interestingly, although catalase did not block vanadate-induced MAPK
activation, it eliminated the vanadate-mediated increase in FasL
production (Fig. 7A). In
contrast, D-JNKI1 and other ROS scavengers/promoters did not
significantly alter FasL production (Fig. 7A). The levels of
FasL were also unaffected by PD98059 (data not shown). These results
indicated that the H2O2 was critical in
vanadate-induced increase in FasL expression.

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Fig. 6.
Effects of catalase, D-JNKI1, and PD98059 on
c-Jun and ERK activation. Cells were pretreated with catalase
(2000 units/ml), PD98059 (50.0 µM), or D-JNKI1 (1.0 µM) for 30 min before vanadate treatment (25.0 µM). A, c-Jun activation. Cells were then
exposed to vanadate for 6 h, and c-Jun activation was determined
as described in the legend for Fig. 2. B, ERK activation.
Cells were then exposed to vanadate for 30 min, and ERK activation was
determined as described in the legend for Fig. 2.
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Fig. 7.
Effects of ROS scavengers and inhibitors for
MAPKs on vanadate-induced FasL production, Fas-FADD association, and
caspase-8 activation. Cells were pretreated with catalase (2000 units/ml), PD98059 (50.0 µM), or D-JNKI1 (1.0 µM) for 30 min before vanadate treatment (25.0 µM). A, FasL expression. Cells were exposed to
vanadate for 24 h, and the expression of FasL was determined by
immunoblot. B, Fas-FADD association. Cells were then exposed
to vanadate for 6 h, and Fas-FADD association was determined as
described in Fig. 3. Panels on the right were the
microdensitometrical quantification of relative expression.
C, caspase-8 activation. Cells were exposed to vanadate for
24 h, and the activation of caspase-8 was determined as described
under "Experimental Procedures." The experiments were replicated
three to four times. *, statistically significant difference relative
to control cultures (p < 0.05); #, statistically
significant difference relative to vanadate-treated cultures
(p < 0.05); @, statistically significant
difference relative to vanadate- and catalase-treated cultures
(p < 0.05); &, statistically significant difference
relative to vanadate and JNKI-treated cultures (p < 0.05).
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Because catalase blocked the vanadate-induced increase in FasL
expression, we examined the effect of catalase on Fas aggregation and
Fas-FADD association. As shown in Figs. 3C and
7B, catalase produced a modest inhibition on
vanadate-induced Fas aggregation and Fas-FADD association. Despite its
lack of effect on FasL production, D-JNKI1 dramatically
inhibited vanadate-induced Fas aggregation and Fas-FADD association.
The combination of catalase and D-JNKI1 treatment completely abolished
vanadate-mediated Fas-FADD association (Fig. 7B). PD98059
did not affect the interaction between Fas and FADD. Taken together,
these observations indicate that vanadate-induced FasL production is
independent of MAPK activation but is dependent on
H2O2 generation. In contrast, JNK activation is
not required for vanadate-induced increase in FasL production but is
essential for the activation of Fas and subsequent Fas-FADD association.
Because vanadate-induced JNK activation triggered Fas-FADD association,
we further examined whether the activation of caspase-8 was mediated by
JNKs. As shown in Fig. 7C, D-JNKI1 abolished
vanadate-induced caspase-8 activation. On the other hand, catalase
produced only a marginal inhibition on vanadate-induced caspase-8
activation. PD98059 had no effect on caspase-8 activation.
Vanadate-induced Apoptosis Is Mediated by FADD-Caspase-8
Pathway--
Because vanadate increased FasL expression (Fig.
3A), we sought to determine whether the apoptosis of
CGPs is mediated by the FasL-Fas interaction. Two Fas antagonists,
SM1/23 and Fas-Fc, were introduced. These antagonists effectively
abolished FasL-Fas interaction (67, 68) and eliminated recombinant
FasL-induced apoptosis in CGPs (data not shown). However, both
antagonists produced only a modest but statistically significant
(p < 0.05) inhibition of vanadate-induced apoptosis
(Fig. 8). These results suggested that
although FasL induction was involved it was not the primary mechanism
for vanadate-induced apoptosis of CGPs.

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Fig. 8.
Effects of Fas antagonists on
vanadate-mediated apoptosis. CGPs were exposed to vanadate
for 24 h with or without antibody SM1/23 (3.0 µg/ml) or Fas-Fc
fusion protein (40.0 µM). Apoptosis was determined
with a DNA fragmentation ELISA. The experiment was replicated four
times. *, statistically significant difference relative to control
cultures (p < 0.05); #, statistically significant
difference relative to vanadate-treated cultures (p < 0.05).
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The Fas-mediated apoptotic pathway is initiated by the recruitment of
FADD followed by the cleavage and activation of caspase-8 (61). To
examine whether vanadate-induced cell death is mediated by the
FADD-caspase-8 pathway, we used DN-FADD and the caspase-8 inhibitor,
Z-IETD-FMK, to block the action of FADD and caspase-8, respectively. As
shown in Fig. 9, both DN-FADD and
Z-IETD-FMK eliminated vanadate-induced cell death. Blockage of the
activation of caspase-3 but not caspase-9 also significantly inhibited
vanadate-induced apoptosis (Fig. 9B). These results
support the notion that the Fas-FADD-caspase-8 pathway plays a major
role in vanadate-triggered cell death in CGPs.

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|
Fig. 9.
Effects of dominant negative FADD
(DN-FADD) and caspase inhibitors on vanadate-mediated
apoptosis. A, cells were transfected with pEGFP-C3 and
DN-FADD or empty pcDNA3.0 vector. One day after transfection cells
were exposed to vanadate (25.0 µM) for 24 h, and
apoptosis in the transfected cells was assayed by nuclei fragmentation
and condensation after TOTO-3 iodide staining as described under
"Experimental Procedures." Relative apoptosis in the total
transfected cells (green fluorescent protein-positive) was quantified.
B, cells were pretreated with different caspase inhibitors
for 30 min before vanadate exposure (25.0 µM, 24 h).
After vanadate treatment, apoptosis was determined with a DNA
fragmentation ELISA. The inhibitors for caspase-3 (Cas3-I),
caspase-8 (Cas8-I), and caspase-9 (Cas9-I) were
Ac-DMQD-CHO, Z-IETD-FMK, and ZlEHD-FMK, respectively. Each data point
(bars; ± S.E.) is the mean of three to four independent
trials. *, denotes a statistically significant difference relative to
control group (p < 0.05). #, statistically significant
difference relative to vanadate-treated cultures (p < 0.05); @, statistically significant difference relative to vanadate
and caspase 3-I-treated cultures (p < 0.05).
|
|
To further investigate the role of JNK activation and ROS in
vanadate-induced apoptosis, we determined whether blockage of MAPK
activation or H2O2 removal could protect CGPs
from vanadate-induced cell death. Removal of
H2O2 by catalase produced a partial and statistically significant (p < 0.05) rescue (Fig.
10). Inhibition of JNK activation
exerted a more potent protection against vanadate-induced damage.
Furthermore, simultaneous treatment with catalase and D-JNKI1
completely abolished vanadate-mediated cell death. Vanadate-triggered cell death remained unaffected by both PD98059 and SB 292190 treatment.

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|
Fig. 10.
Effects of inhibitors for MAPKs and catalase
on vanadate-induced apoptosis. Cells were pretreated with catalase
(2000 units/ml), PD98059 (50.0 µM), SB292190 (10.0 µM), or D-JNKI1 (1.0 µM) for 30 min before
vanadate treatment (25.0 µM; 24 h). After vanadate
exposure, apoptosis was determined with a DNA fragmentation ELISA. Each
data point (bars; ± S.E.) is the mean of four independent
trials. *, statistically significant difference relative to control
cultures (p < 0.05); @, statistically significant
difference relative to vanadate-treated cultures (p < 0.05); #, statistically significant difference relative to
vanadate- and catalase-treated cultures (p < 0.05);
&, statistically significant difference relative to
vanadate- and JNKI1-treated cultures (p < 0.05).
|
|
Vanadate may serve as a PTP inhibitor. To determine whether other
inhibitors for PTP have the same effect as vanadate, we examined the
effect of
-bromo-4-hydroxyacetophenone, a specific PTP inhibitor, on
CGPs. The results indicated that this inhibitor did not significantly
induce apoptosis (data not shown), suggesting that the effect of
vanadate is not mediated by a general inhibition of tyrosine dephosphorylation.
 |
DISCUSSION |
The present study demonstrates that vanadate at physiologically
relevant concentrations (10-100 µM) inhibits the growth
of cultured CGPs. These concentrations, which range in micromoles, are
comparable with the levels encountered in industrial settings (29). The
CGPs proliferate in vitro, and vanadate produces a marginal
inhibition of the proliferation of CGPs. On the other hand, vanadate
dramatically increases the death of CGPs. Therefore, vanadate-induced
growth inhibition mainly results from cell death. Vanadate-induced
death of CGPs is typical of apoptosis, which is verified by measuring
various apoptotic indices. Furthermore, the present study characterizes
a signaling pathway that explains how vanadate exposure induces
neuronal apoptosis.
In general, the apoptotic signal can be transmitted through either the
death receptors, such as Fas, or mitochondria (63). Fas (also called
APO-1 or CD95) is a member of the tumor necrosis receptor superfamily
that transmits a death signal to the cells (61). In Fas-mediated death,
it is well known that the binding to its ligand, FasL, or agonist
antibodies induces aggregation of the receptor, which results in its
interaction with the death adapter molecule Fas-associated death domain
(FADD, also called MORT1) (63). FADD contains a death domain (DD) and a
death effector domain (DED). On activation, Fas associates with the
death domain in FADD (61). Subsequently, procaspase-8 binds to the DED
of FADD and forms a death-inducing signal complex (DISC). on forming a
DISC, procaspase-8 is activated autoproteolytically and propagates the
apoptotic signal by activating executioner caspases such as caspase-3
(63, 69, 70). Formation of the DISC could be FasL-dependent or independent (51-74). In mitochondria-mediated cell death,
mitochondria sense the apoptotic signal and convey it to the activation
of adapter APAF-1 via the release of cytochrome c.
Cytochrome c binds to APAF-1, and in the presence of adenine
nucleotides the APAF-1-cytochrome c complex promotes
activation of procaspase-9 (75). Caspase-9 can activate executioner
caspases and trigger apoptosis (63).
Vanadate clearly triggers a death signal mediated by the death receptor
Fas. It up-regulates FasL expression, promotes the Fas aggregation and
the association of Fas with FADD, and subsequently activates caspases
(caspase-8 and -3). Apparently, vanadate-induced apoptosis occurs
through this pathway because blocking this pathway by either a DN-FADD
or caspase-8 inhibitor eliminates vanadate-induced cell death. In
Fas-mediated apoptosis, Fas is activated first by the binding to its
ligand (FasL) or agonist antibody (61). Our results show that vanadate
induces a robust increase of FasL expression. Surprisingly, it appears
that the FasL induction has a relatively minor contribution to
vanadate-induced apoptosis. This argument is based on the observations
that (a) the elimination of vanadate-induced increase in
FasL expression (by treatment of catalase) does not inhibit Fas-FADD
association, and only produces a modest protection against
vanadate-induced apoptosis, and (b) a blockage of
FasL-Fas interaction by Fas antagonists has a marginal effect on
vanadate-induced cell death. Thus, it can be concluded that, although
it participates, FasL induction is not the major contributor to
vanadate-induced apoptosis. This may be the result of the inefficiency
in the level of FasL induction to cause a substantial cell death.
Therefore, there must be other mechanisms independent of FasL induction
that account for vanadate-induced death of CGPs.
Vanadate activates two MAPKs, ERK1/2 and JNK1, in CGPs. The pattern of
activation for each is different. The activation of ERKs is rapid and
transient, whereas JNK activation is relatively slow and more
persistent (lasting for at least 12 h). In addition, vanadate
induces ROS generation in CGPs. It appears that
H2O2 is the major precursor of the ·OH
radical induced by vanadate because catalase (a specific
H2O2 scavenger) eliminates the ·OH
radical generated by vanadate. It has been demonstrated that many
effects of vanadate are mediated by its ability to generate ROS. For
example, vanadate-induced activation of transcription factors such as
NF-
B, p53, or hypoxia-inducible factor 1
(HIF-1
) and the
up-regulation of tumor necrosis factor
(TNF
) expression are
clearly mediated by ROS generation, particularly through the H2O2 accumulation (76-79). Vanadate-induced
JNK activation has been observed in mouse macrophages, and the
activation is also dependent on ROS induction (76). However, the
present study demonstrates that the H2O2
scavenger catalase does not affect the activation of JNK and ERKs,
suggesting that ROS is not involved in vanadate-induced
activation of MAPKs in CGPs. Vanadium compounds may serve as a PTP
inhibitor that enhances protein tyrosine phosphorylation (59, 60). Our
results show that vanadate increases tyrosine phosphorylation on JNK1,
which suggests that vanadate-induced activation of signaling proteins
is mediated by its inhibition of PTPs. Thus, it appears that the
biological effect of vanadate is cell type-specific.
JNK activation can result in FasL expression in various cells (22,
80-82). However, the present study shows that neither JNK nor ERK
activation is required for vanadate-induced increase in FasL production
in CGPs. On the other hand, ROS appears to be indispensable for
vanadate-induced FasL production; the H2O2 scavenger catalase completely blocks vanadate-induced FasL expression. The exact signal pathway underlying ROS regulation of FasL expression in CGPs remains to be elucidated. Although JNK activation is not involved in FasL induction, it is required for vanadate-induced Fas-FADD association. That is, vanadate-induced JNK activation initiates the Fas-FADD-caspase pathway in a FasL-independent manner. This effect is mediated by FasL-independent Fas aggregation. It has
been reported that JNK activation triggers FasL-independent aggregation
of Fas and subsequently evokes the FADD-caspase pathway in Jurkat T
cells (73). FasL-independent Fas aggregation has been observed on
various stress stimuli, such as exposure to UV, cycloheximide,
cisplatin, etoposide, vinblastine, curcumin, and doxorubicin
(83-87).
Persistent activation of JNK is capable of triggering apoptotic
pathways initiated by both the death receptor (such as Fas) and
mitochondria (88). Apoptosis of CGPs caused by vanadate-induced JNK
activation is mediated primarily by the death receptor Fas. This
conclusion is supported by the following observations. First, vanadate-induced apoptosis is mediated by the FADD-caspase-8 pathway, and the blockage of JNK activation abolishes Fas-FADD association and
caspase-8 activation. Second, inhibition of JNK activation significantly reduces vanadate-mediated cell death. FasL induction has
a modest but statistically significant contribution to vanadate-induced apoptosis. Simultaneously blocking JNK activation and FasL expression totally abolishes vanadate-induced Fas-FADD association and the subsequent apoptosis. This result indicates that both JNK activation and FasL induction contribute to vanadate-induced apoptosis, and JNKs
are the major contributors. Third, vanadate activates caspase-8 and -3 but not caspase-9. Fas activation (Fas aggregation) results in the
formation of DISC that activates procaspase-8 (61). Activated caspase-8
directly cleaves procaspase-3, an executioner caspase, and triggers
apoptosis (89). On the other hand, the activation of caspase-9 is
triggered mainly by damage to mitochondria and the release of
cytochrome c. It is noted, however, that cross-talk may
exist between death receptor- and mitochondria-mediated cell death (90,
91).
In summary, JNK activation by vanadate initiates the Fas-FADD-caspase-8
signal pathway and triggers apoptosis in CGPs. The finding that
vanadate induces apoptosis in central nervous system neurons may
explain some of the central nervous system defects attributed to
environmental exposure to vanadate. The JNK and FADD-caspase-8 signal
pathways may also contribute to the neurotoxicity of other
environmental toxicants, such as heavy metals. Like vanadate, the heavy
metals, including arsenite, lead, lithium, and mercury, have been shown
to induce neuronal apoptosis (26, 71, 92-93). Understanding the role
of JNK in apoptosis of neuronal precursors will help elucidate the
mechanisms of programmed cell death in the developing nervous system.
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M208295200
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
BrdUrd, 5-bromo-2'-deoxyuridine;
CGPs, cerebellar granule progenitors;
DISC, death-inducing signaling
complex;
ERK, extracellular signal-regulated kinase;
FADD, Fas-associated death domain;
DN-FADD, dominant negative FADD;
FasL, Fas
ligand;
JNK, c-Jun NH2-terminal kinase;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
MEK, MAPK/ERK kinase;
PBS, phosphate-buffered saline;
PTP, protein tyrosine
phosphatase;
ROS, reactive oxygen species;
ELISA, enzyme-linked
immunosorbent assay;
DMPO, 5,5-dimethyl-1-pyrroline N-oxide;
DISC, death-inducing signal complex;
APAF-1, apoptotic
protease-activating factor-1.
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