From the Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel
Aviv 69978 Israel, the § Department of Biochemistry and
Biophysics, Medical Nobel Institute for Biochemistry, Karolinska
Institute, S-171 77 Stockholm, Sweden, the ¶ Department of
Neurology and Felsenstein Medical Research Institute, Rabin Medical
Center and Sackler School of Medicine, Tel Aviv University, Ramat Aviv,
Tel Aviv 69978 Israel, and the
Interdepartmental Core Facility,
Sackler School of Medicine, Tel Aviv University, Ramat Aviv,
Tel Aviv 69978 Israel
Received for publication, September 6, 2000
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ABSTRACT |
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The neurotransmitter dopamine (DA) induces
apoptosis via its oxidative metabolites. This study shows that
glutaredoxin 2 (Grx2) from Escherichia coli and human
glutaredoxin could protect cerebellar granule neurons from DA-induced
apoptosis. E. coli Grx2, which catalyzes
glutathione-disulfide oxidoreduction via its -Cys-Pro-Tyr-Cys- active
site, penetrates into cerebellar granule neurons and exerts its
activity via NF- The thiol-disulfide metabolism and balance contributes to the
maintenance of the cellular redox (reduction/oxidation) state. Thiol
redox control (1) can affect a protein's synthesis and folding, the
assembly into multimeric complexes, the enzymatic activity or the
binding activity of transcription factors (Refs. 2 and 3; for review,
see Ref. 4). Intracellular redox-active molecules such as thioredoxin,
glutaredoxin, and Ref-1, and low molecular weight thiols such as
glutathione (GSH), distribute and maintain a reduced cytosolic
environment in the normal cell.
Dopamine (DA),1 the
endogenous neurotransmitter of the nigro-striatal pathway, is a
powerful oxidant that exerts its toxic potential through its oxidative
metabolites. Administration of DA into the rat striatum (5) caused pre-
and postsynaptic damage. Intraventricular injection of DA into rats
resulted in dose-dependent death of the animals (6).
In vitro studies have shown that DA can cause cell death in
mesencephalic, striatal, and cortical primary neuron cultures (7-11).
Moreover, we have shown that DA-induced cell death in sympathetic,
cerebellar granule neurons, PC-12 cells, and thymocytes has all the
characteristic features of apoptotic cell death (12-14).
Glutaredoxins (Grx) are generally 10-kDa proteins, which catalyze
GSH-disulfide oxidoreductions via two redox-active cysteine residues
(15). The active site sequence (Cys-Pro-Tyr-Cys) is conserved in a
variety of species (16-23). Glutaredoxins can reduce either
intramolecular disulfides as in the case of ribonucleotide reductase
(24-26), or mixed disulfides between a thiol-containing compound and
GSH (25, 27). Escherichia coli contains three glutaredoxins, with Grx2 being the predominant in terms of
concentration and catalytic activity (80% of total GSH-oxidoreductase
activity in E. coli) (28). Currently, only one human
glutaredoxin has been reported (23). The protein is generally
considered intracellular but may also be found in human plasma (29).
Apart from its activity with ribonucleotide reductase, human
glutaredoxin can reduce the GSH-mixed disulfide of inactive oxidized
nuclear factor-I and thus restore its DNA binding activity (30).
Glutaredoxin has a role in reduction of mixed disulfides in cells
exposed to oxidative stress (31).
The nuclear factor- Ref-1 regulates the activity of several transcription factors,
including AP-1 elements (Jun-Fos dimers) and NF- The data presented here demonstrate that E. coli and human
glutaredoxins could protect cerebellar granule neurons from DA-induced death by activating the DNA binding activity of NF- Postnatal day 8 BALB/c mice were obtained from Tel Aviv
University Animal Care Facility (Glasberg Animal Research Tower). Culture media, sera, and trypsin (0.25% in 0.05% EDTA) were from Biological Industries Co. (Beit Hemeek, Israel). 32P was
obtained from PerkinElmer Life Sciences, Sephadex G-25 from Amersham Pharmacia Biotech (Uppsala, Sweden), and NF- Primary Culture of Cerebellar Granule Neurons--
Cultures of
highly enriched granule neurons were obtained from postnatal day 8 BALB/c mice (52). Cells were dissociated by trypsinization and plated
in standard medium (basal Eagle's medium, 10% fetal calf serum, 25 mM KCl, 2 mM glutamine, 50 µg/ml gentamycin,
and 250 ng/ml amphotericin B, supplemented with 1 mg/ml glucose (53) on
dishes coated with poly-L-lysine (cell density, 7 × 106 cells/35-mm diameter dish, 1.5 × 106
cells/well for a 24-well plate, 2.5 × 105 cells/well
for a 96-well plate). Cytosine- Treatments--
DA (3-hydroxytyramine hydrochloride, CH8502,
Sigma) was dissolved directly in the proper culture medium. Cerebellar
granule neurons were maintained in standard medium for 6-7 days. The
medium was then replaced with serum-free standard medium with DA,
rhGrx, Grx2, or Grx2 mutants, for various periods of time. Control
cultures were maintained in serum-free standard medium.
Prior to addition to the neurons, glutaredoxins were reduced with 2 mM DTT for 20 min at 37 °C. DTT was removed by two spin columns of Sephadex G-25 equilibrated with phosphate-buffered saline
(PBS).
Analysis of Neuronal Viability--
The viability of the
cultures (96-well plates) after treatment with DA and/or glutaredoxins
was assessed either by Alamar Blue assay (AccuMed) or by
4,6-diaminodiphenyl-2-phenylindole (DAPI) staining. Alamar reagent
(1:10) was added to the treated cells for 2-2.5 h at 37 °C. The
viability was evaluated by subtracting the fluorescence of the medium
alone (without cells) from the fluorescence of the cells at 530 nm
excitation wavelength and 590 nm emission wavelength. Cells were
fixated with 4% paraformaldehyde for 30 min, rinsed with PBS, and
incubated with 5 µg/ml DAPI for 5 min. Nuclei were visualized under
UV light. Each assay was carried out in triplicate.
Determination of the Apoptotic Commitment Point--
Neurons
were treated with 600 µM DA. At different time points
following DA exposure (1-24 h), the drug was washed out and replaced
with fresh medium without DA. Similar sets of untreated cells served as
control. Cell survival was monitored 24 h after DA administration,
as mentioned above.
Protein Content--
Protein cell content was determined
according to the method of Bradford (55) using bovine serum albumin as standard.
Immunocytochemistry and Nuclear Staining with
DAPI--
Cerebellar granule neurons were grown on glass coverslips
coated with poly-L-lysine hydrobromide (P2636,
Sigma).Treated neurons were fixed for 30 min in ice-cold 4%
paraformaldehyde/PBS, washed with PBS (pH 7.4), permeabilized, and
blocked for 20 min with 0.2% Triton X-100 in 10% normal goat serum.
Cells were then incubated with anti-p65 or anti-Ref-1 antibodies (1:50)
in PBS containing 2% normal goat serum for 1 h at 37 °C,
washed, and then incubated for 1 h with Cy3- or Cy2-conjugated
secondary antibody (Jackson Laboratories, Bar Harbor, ME). To identify
the cellular location of Ref-1 or p65, the DNA of the cells were
stained with DAPI.
Protein
Biotinylation--
Sulfosuccinimidyl-6-(biotinamido)hexanoate (catalog
no. 21335) (biotin) was purchased from Pierce. Grx2 and C9S-C12S mutant were incubated with biotin (75 µg of biotin/mg of protein) in PBS
buffer for 16 h at 4 °C. Residual biotin was removed using Sephadex G-25 column before being given to cultures. Biotinylation was
confirmed by Western blot analysis using streptavidin-horseradish peroxidase.
Biotinylated Grx2 and C9S-C12S mutant were incubated for 30 min with
the neurons. Thereafter, the slides were washed, fixed for 5 min in
methanol (
To prevent bleaching of the fluorescence, moviol with
n-propyl gallate was added to the slides before being
excited with fluorescein and rhodamine filters.
Treatment Affecting Coated Pit Structure--
Hypertonic
treatment, which blocks endocytosis via coated pits by dispersing the
underlying clathrin lattices (55), was performed by incubating the
culture for 15 min in PBS supplemented with 0.45 M sucrose.
The cells were kept in hypertonic medium during incubation with Grx2.
Confocal Microscopy--
Fluorescently-stained cells were
analyzed using Zeiss confocal laser scanning microscope. Zeiss LSM 410 inverted (Oberkochen, Germany) is equipped with a 25-milliwatt
krypton-argon laser (488 and 568 maximum lines) and 10-milliwatt
helium-neon laser (633 maximum lines). A 40× numeric aperture/1.2 C
Apochromat water-immersion lens (Axiovert 135M, Zeiss) was used for all imaging.
Western Blot Analysis--
Western blot analysis was performed
as described by Harlow and Lane (57), using 12.5% polyacrylamide gels.
Each lane was loaded with an equal amount of protein extracts (30 or 60 µg), which, following electrophoresis, was transferred to an
Immobilon polyvinylidene difluoride membrane for 1.5 h. Blots were
stained with Ponceau to verify equal loading and transfer of proteins. Membranes were then probed with anti-Ref1 (Santa Cruz Biotechnology), anti-I Nuclear and Cytoplasmic Extracts--
Neurons (7 × 106 cells) were washed with phosphate-buffered saline,
scraped off, pelleted, and resuspended in 30 µl of hypotonic buffer A
(10 mM Tris-HCl (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 1 mM DTT, and
protease inhibitors). After 15 min on ice, Nonidet P-40 was added
(0.6%) and the lysates were spun down at 14,000 rpm at 4 °C.
The supernatant was removed (cytoplasmic extract), and the nuclear
pellet was resuspended in 20 µl of buffer B (20 mM
Tris-HCl (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, and protease inhibitors) with frequent vortexing
for 30 min at 4 °C. Finally, the nuclear extract was spun at 14,000 rpm for 10 min and the supernatant was used for electrophoretic
mobility shift assays (EMSA).
EMSA--
The binding reaction mixture containing 10 mM Tris-HCl (pH 7.9), 60 mM KCl, 0.4 mM dithiothreitol, 10% glycerol, 2 µg of bovine serum
albumin, 1 µg of poly(dI-dC), 15,000 cpm of 32P-labeled
Antisense Oligonucleotide Treatment--
Ref-1 antisense
oligonucleotide and the complementary sense oligonucleotide were
synthesized and high performance liquid chromatography-purified by
Sigma Genosys Ltd. The oligonucleotides were labeled with fluorescein at the 5' end and phosphorotioated at the 3' end (at the positions marked by *) to confer nuclease resistance. The sequence of the Ref-1
antisense probe was 5'-TTCCCCGCTTTGGCAT*C*G*C-3' and the sense
5'-GCGATCCCAAAGCGGGGAA-3' (nucleotides
The oligonucleotides were dissolved (10 mM stock) in
distilled water prior to use and diluted to a final concentration of 5 and 10 µM in serum-free medium. Cerebellar granule neuons
were treated with oligonucleotides 6 days after plating.
Cotransfection of Cultured Cerebellar Granule
Neurons--
Cerebellar granule neurons (1.5 × 106)
were transfected 6 days after plating using the calcium phosphate
method (58). Cells were cotransfected with a plasmid encoding green
fluorescent protein (GFP) and with pl I- Glutaredoxin Activity Protects Neuronal Cells from DA- induced
Deaths (Fig. 1)--
To determine
whether glutaredoxin could protect neuronal cells from DA-induced
death, cerebellar granule neurons were exposed to 600 µM
DA for 5 h, in the presence of various concentrations (2-13.8
µM) of reduced E. coli Grx2, Grx2 mutants C12S
(exhibits 70% of the wild type activity) and C9S-C12S (inactive) and
recombinant human glutaredoxin (rhGrx) (Fig. 1). In the absence of
Grx2, only 20% of the exposed neurons survived. Increasing
concentrations of wild type Grx2 rescued exposed neurons from apoptotic
death (84%) (Fig. 1A). The protective effect was lower for
the Grx2 mutant C12S (52%), whereas no protection was conferred by the double thiol mutant (Grx2 C9S-C12S) (Fig. 1A). rhGrx showed
an almost identical protection profile as E. coli Grx2 (Fig.
1B). Counting condensed and apoptotic nuclei using DAPI
staining revealed that Grx2 (4-13 µM) treatment
conferred full protection against DA toxicity (Fig. 1C).
These results show that glutaredoxins (E. coli and human)
protected neurons from DA-induced death. The redox activity of Grx2 was
essential for its protective effect.
Grx2 Penetrates into the Granule Neurons (Fig.
2)--
To examine whether Grx2 could
penetrate neurons, neurons were incubated with 13 µM Grx2
for 30 min, fixed, and exposed to anti Grx2 antibodies. No
cross-reacting material was detected (data not shown). After incubating
neurons with biotinylated Grx2, cross-reacting material was observed
throughout 100% of the cells (cytoplasm and nucleus, Fig.
2C). No signal was obtained from samples in which
biotinylated Grx2 was omitted (Fig. 2A). To exclude the
possibility that the streptavidin binds to residual levels of free
biotin and not to biotinylated Grx2, the neurons were exposed to biotin
eluted from Sephadex column (similar to biotinylated proteins), fixed,
and then reacted with labeled streptavidin. No signal was obtained
under these conditions (data not shown). These results demonstrate that
small amounts of wild type Grx2 could penetrate into neurons. Since the
inactive double thiol mutant C9S-C12S could also be detected
intracellularly (Fig. 2E), it is most likely that the
penetration of Grx2 is independent of the redox activity of the
protein.
To examine whether coated pit vesicles were involved in Grx2
endocytosis, neurons were exposed to biotinylated Grx2 in the presence
of 0.45 M sucrose, which disperses coated pit structures. An indistinguishable pattern of Grx2 permeabilization was observed in
the presence or absence of sucrose, suggesting that Grx2 penetrates neurons via mechanisms other than those of coated pit vesicles (data
not shown).
DA Decreases whereas Grx2 Increases
NF-
NF-
Exposure of cerebellar granule neurons to DA for 1 h did not
affect the binding activity of NF-
The Grx2 C12S monothiol mutant activated slightly the DNA binding
activity of NF- Grx2 Affects Both the Translocation of NF-
Next we examined whether Grx2 was affecting the translocation of
NF- NF-
We next examined whether inhibition of NF- Grx2 Regulates NF-
Since the levels of Ref-1 and NF- DA, the endogenous neurotransmitter of the nigro-striatal pathway,
is a powerful oxidant that exerts its toxic potential through its
oxidative metabolites. Reducing thiols such as DTT, NAC, and GSH are
capable of neutralizing DA oxidative metabolites and thereby conferring
protection against DA-induced apoptosis (14). In the search of more
physiological thiol antioxidant, we found that E. coli Grx2
and human glutaredoxin could protect neuronal cells against DA-induced
death by activating the NF- Next, we looked for the intracellular targets of Grx2. Transcription
factors whose DNA binding activity is redox-dependent were
good candidates (reviewed in Ref. 4). Such transcription factors are
the NF- The fact that Grx2 stimulates NF- Human and E. coli Grx2 differ significantly in their
catalytic activity using the mixed disulfide between the
Monothiol Grx2 could protect neurons from DA-induced
apoptosis, but to a lesser extent than the dithiol wild type
glutaredoxin. Known glutaredoxins can reduce a disulfide substrate
through a monothiol or a dithiol mechanism (73). For the reduction of protein disulfides, both cysteine residues (dithiol mechanism) are
required. A single amino-terminal cysteine can specifically reduce
protein-GSH mixed disulfides (monothiol mechanism; Refs. 26 and 74). In
the latter case, GSH forms a mixed disulfide with glutaredoxin
(GrxS-SG), while the protein component is released in its reduced form.
The GrxS-SG can be reduced by GSH to give reduced glutaredoxin and
oxidized glutathione (GSSG). The ability of monothiol Grx2 to confer
increased DNA binding of NF- The contribution of NF- Recently, Nakamura et al. (29) showed that Grx and Trx are
found in human plasma, and suggested that they may be part of plasma
defense mechanisms against oxidative stress. We propose that, besides
being part of the plasma defense mechanism, redox-active glutaredoxins
function as a signal molecule that penetrates the cells and activates
signal pathways by affecting redox regulation.
In summary, our results demonstrate that Grx2 protects cerebellar
granule neurons from DA-induced apoptosis by activating NF-B activation. Analysis of single and double cysteine
to serine substitutions in the active site of Grx2 showed that both
cysteine residues were essential for activity. Although DA
significantly reduced NF-
B binding activity, Grx2 could stimulate the binding of NF-
B to DNA by: (i) translocating NF-
B from the cytoplasm to the nucleus after promoting the phosphorylation and degradation of I-
B
, and (ii) activating the binding of pre
existing nuclear NF-
B. The DNA binding activity of NF-
B itself
was essential for neuronal survival. Overexpression of I-
B dominant
negative gene (I-
B-
N) in granule neurons significantly reduced
their viability, irrespective of the presence of Grx2. Ref-1 expression was down-regulated by DA but up-regulated by Grx2, while treatment of
neurons with Ref-1 antisense oligonucleotide reduced the ability of
Grx2 to activate NF-
B binding activity. These results show that Grx2
exerts its anti apoptotic activity through the activation of Ref-1,
which then activates NF-
B.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B NF-
B/Rel is one of the transcription factors
whose DNA binding activity is regulated by a redox mechanism. Agents
that modify free sulfhydryls such as N-ethylmaleimide and diamide inactivate the DNA binding of NF-
B, whereas reductants such
as dithiothreitol (DTT),
-mercaptoethanol, and thioredoxin enhance
its DNA binding activity (for review, see Ref. 4; Ref. 32). NF-
B
regulates the expression of various genes related to the immune
response, stress, inflammation, and the inhibition of apoptosis (33,
34). Ordinarily, NF-
B proteins are expressed in an inactive form
bound to an inhibitory protein referred to as I-
B. Following
appropriate stimuli (UV radiation, inflammatory cytokines, phorbol
esters, reactive oxygen species (ROS)), NF-
B is released from the
cytoplasmic complex by phosphorylation and ubiquination-dependent degradation of I-
B. The
I-
B-released NF-
B dimer translocates into the nucleus, where it
binds to cognate DNA sequences and activates transcription of specific
target genes (35-37). NF-
B is activated in a number of in
vivo model systems of brain injury: brain trauma, focal ischemia,
and kainate-induced seizure (38-41). Immunohistochemical analysis of
brain sections from Alzheimer's disease patients revealed that NF-
B
was activated in the most damaged areas of the brain (42). In
Parkinson's disease patients, the number of neurons with
NF-
B-stained nuclei was 70-fold higher than that of control
subjects, suggesting that translocation of NF-
B to the nucleus was
related to the pathophysiology of the disease (43).
B. In addition, Ref-1 possesses apurinic/apyrimidinic endonuclease DNA repair activity
against DNA damage caused by ROS, UV, and IR radiation (44-46). It is
expressed in subpopulations of cells in the brain, including granule
neurons of the cerebellum (47, 48). Ref-1 itself is subject to redox
control and was shown to interact with thioredoxin (49).
B through Ref-1.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B inhibitor (SN50) from Promega (Madison, WI). Rabbit antibodies against NF-
B p65 and Ref-1 were purchased from Santa Cruz Biotechnology Inc (Santa
Cruz, CA). I-
B-
N plasmid was a gift from Prof. Yinon Ben-Neriah.
E. coli Grx2 was a recombinant preparation purified to
homogeneity as described (50). The single and double cysteine to serine
mutants in the active site of Grx2 were prepared as described
previously (50), but without His tags, and purified to homogeneity with
the method for the wild type protein. Antibodies to Grx2 were raised in
a rabbit by standard techniques. Recombinant human Grx was prepared as
described by (51).
-arabinofuramoside (Ara-C) (10 µM) was added to the medium 18-22 h after plating to
prevent replication of non neuronal cells (54).
20 °C) followed by a 2-min incubation in acetone
(
20 °C), and blocked with 1% bovine serum albumin and goat
-globulin (Jackson Laboratories) (200 µg/ml) for 1 h. Next,
the slides were washed and incubated with Cy3-conjugated streptavidin
(0.5 µg/ml, Jackson Laboratories) for 30 min. After overnight washing
in PBS, the slides were mounted.
B
or anti-phospho-I
B
(Ser32) antibodies (New
England Biolabs) (1:1000). Intensity of the signal was determined by
ECL-Plus detection system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).
B oligonucleotides (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega, Madison,
WI) was incubated for 30 min with 5 µg of nuclear extract. For
specificity control, a 50-fold excess of unlabeled probe was applied.
In the antibody supershift assay, the reaction mixture minus probe was
incubated with 2 µg of p50 and/or p65 antibodies (Santa Cruz
Biotechnology) for 15 min at room temperature. Products were analyzed
on a 5% acrylamide gel made up in 1× TGE (50 mM Tris, 400 mM glycine, 2 mM EDTA). Dried gels were exposed
to x-ray film or to phosphor screen (Molecular Dynamics). Quantitative data were obtained using PhosphorImager analysis (Molecular Dynamics).
3 to 16). The probe did not
show homology to other known genes according to the GenBankTM data base.
B-
N gene
(pRC-CMV-I-
B-
N), or with the GFP plasmid and the pRC-CMV empty
vector. Cotransfections were carried out using a ratio of 1:1. 24 h after transfection, the medium was replaced by serum-free medium with
or without DA and/or Grx2, and neuronal viability was analyzed by
counting GFP positive cells under microscope (×20). For each
treatment, four different fields from four different wells were analyzed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Neuroprotective activity of Grx2, Grx2
mutants, and rhGrx against DA-induced death. Cerebellar granule
neurons were incubated with 600 µM DA for 5 h in the
presence or absence of various concentrations of Grx2 ( ), C9S mutant
(
), or C9S-C12S mutant (
) (A) or rhGrx (B).
Cell viability was determined by Alamar blue assay or by counting
condensed and apoptotic nuclei as shown by DAPI staining
(C). ***, p < 0.001; **, p < 0.025; *, p < 0.05. Error
bars represent ± S.D. Statistical analyses were
performed with two-tailed Student's t test
(n = 4).
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Fig. 2.
Biotinylated Grx2 and C9S-C12S mutant
penetrate into the granule neurons. Cerebellar granule neurons
were incubated for 30 min with 13 µM biotinylated Grx2 or
C9S-C12S mutant, washed, fixed, reacted with Cy3-conjugated
streptavidin, and analyzed by confocal microscopy. A, cells
reacted with Cy3-conjugated streptavidin only (without biotinylated
proteins). C and E, detection of intracellular
Grx2 and C9S-C12S mutant, respectively. B, D, and
F, Nomarski imaging of the cells presented in A,
C, and E, respectively. Bar indicates
10 µm.
B Binding Activity in Cerebellar Granule Neurons (Figs.
3 and
4)--
To determine the duration of
exposure to DA required to trigger the apoptotic process, neurons were
exposed to DA for various incubation times, followed by removal of DA
and re-addition of serum-free medium. Neuronal viability was assessed
after 24 h. Cells recovered well after exposure to DA for 1 h. 50% of the neurons did not recover after 1.5-2 h of exposure (data
not shown), which was therefore set as the commitment time for
apoptosis.
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Fig. 3.
NF- B binding
activity in cerebellar granule neurons following exposure to DA and/or
Grx2. A, EMSA showing NF-
B binding activity in
nuclear extracts from cerebellar granule neurons treated with 600 µM DA, or with 8.3 µM Grx2 or cotreated
with both DA and Grx2 for 1 and 2 h. Sister cultures were
incubated in serum-free medium and served as control. To analyze the
specificity of the shifted bands, nuclear proteins were pre-exposed to
50-fold excess of unlabeled NF-
B consensus sequence, followed by
incubation with radiolabeled probe (Competition). The
arrows indicate the positions of the NF-
B-DNA complexes.
B, quantitative analysis of NF-
B induction is represented
as percentage of untreated cells (n = 5). ***,
p < 0.001; **, p < 0.025; *,
p < 0.05. Error bars
represent ± S.D. Statistical analyses were performed with
two-tailed Student's t test.
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Fig. 4.
NF- B binding
activity in cerebellar granule neurons following treatment with wild
type Grx2 and mutants. A, EMSA showing NF-
B binding
activity in nuclear extracts from cerebellar granule neurons treated
with 8.3 µM Grx2, or with C12S monothiol mutant
(partially active), or with C9S-C12S double thiol mutant (inactive).
The first lane shows the NF-
B complexes
isolated from untreated neurons. The arrows indicate the
positions of NF-
B-DNA complexes. B, quantitative analysis
of NF-
B induction is represented as percentage of untreated cells
(n = 4). **, p < 0.025; *,
p < 0.05. Error bars
represent ± S.D. Statistical analyses were performed with
two-tailed Student's t test.
B is a transcription factor known to respond to redox signals and
could thus be a potential signal molecule to mediate Grx2 activity. We
initially examined the DNA binding activity of NF-
B in neurons
exposed to DA and/or Grx2 for 1 and 2 h (2 h being the commitment
time). EMSA revealed three distinct bands (Fig. 3). To examine whether
these bands were specific to NF-
B, nuclear extracts were exposed to
radiolabeled probes in the presence of a 50-fold excess of unlabeled
sequences. All three bands were significantly reduced in the presence
of the unlabeled oligonucleotides, indicating that the three distinct
bands were NF-
B-specific. Supershift analysis revealed that the
upper shifted band corresponds to the p50/p65 heterodimer, the middle
band to the p50/p50 homodimer and the lower band did not contain either
p50 or p65 subunits.
B. Further exposure led to a
significant decrease in the intracellular levels of DNA-bound NF-
B.
On the other hand, incubation of the neurons with Grx2 significantly
augmented the DNA binding activity of NF-
B in a time-dependent manner. Rescuing concentrations of Grx2
could abrogate the DA effect on NF-
B binding.
B (3-fold activation) compared with wild type Grx2
(10-fold activation). The levels of DNA-bound NF-
B remained
unchanged when neurons were incubated with the redox inactive C9S-C12S
Grx2 mutant. These results demonstrate that only redox-active Grx2
could increase the DNA binding activity of NF-
B.
B and Its DNA
Binding Activity (Figs. 5 and
6)--
At least two separate mechanisms
can account for alterations in the binding activity of NF-
B: the
redox state of NF-
B and its translocation from the cytoplasm to the
nucleus. To investigate whether Grx2 was affecting the redox state of
NF-
B, isolated nuclei were exposed to 8.3 µM Grx2 for
30 min at 37 °C. Significant stimulation of NF-
B binding activity
to DNA was observed (Fig. 5). Treatment of nuclear extracts with 1 mM GSH had almost no effect on NF-
B binding activity.
Exposure of isolated nuclei to DA resulted in reduced levels of NF-
B
binding, as observed also in whole cell experiments (Fig. 3). NF-
B
binding activity decreased also after exposure of cerebellar granule
neurons or their nucleic extracts to hydrogen peroxide (30 µM), suggesting that ROS lead to decreased NF-
B DNA
binding activity in those cells (data not shown). DA-induced loss of
NF-
B binding activity was restored by GSH or Grx2. However, Grx2
(8.3 or 16.6 µM) was more efficient than 1 mM
GSH. These results suggest that NF-
B proteins inactivated by
oxidative compounds could be reactivated by reducing agents
(glutaredoxin, GSH). In addition, a portion of the nuclear NF-
B
proteins is not bound to DNA. Upon the appropriate conditions, this
portion can be readily activated by Grx2 and bind to DNA.
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Fig. 5.
Modulation of NF- B
binding activity by oxidizing and reducing agents in isolated nuclear
extracts. A, nuclear proteins were isolated from
neurons grown in serum-containing medium. The binding activity of
untreated nuclear extracts was used as reference (Control).
Isolated nuclear extracts were treated for 30 min at 37 °C with 8.3 µM Grx2, or 1 mM GSH or 600 µM
DA, in the presence or absence of 1 mM GSH, 8.3 or 16.6 µM Grx2 as indicated (n = 4).
B, **, p < 0.025; *, p < 0.05; ··, p < 0.025, between DA-treated neurons
and DA+GSH- or DA+Grx-treated neurons. Error bars
represent ± S.D. Statistical analyses were performed with
two-tailed Student's t test.
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Fig. 6.
Grx2 affects the translocation of
NF- B and its binding to DNA.
A, EMSA showing NF-
B DNA binding activity in nuclear
extracts isolated from cerebellar granule neurons treated with 600 µM DA, 8.3 µM Grx2, cotreated with 600 µM DA + 8.3 µM Grx2, or left untreated.
The second and fourth lanes
show NF-
B activity following addition of 8.3 µM Grx2
to isolated nuclear extracts isolated from control cells or DA-treated
cells, respectively. Note that nuclear levels of NF-
B were
significantly higher in nuclear extracts isolated from Grx2-treated
neurons compared with the maximum NF-
B binding following addition of
Grx2 to the isolated nuclear extract. The same level of NF-
B
activation was found after addition of 8.3 µM or 16.6 µM Grx2 for 30, 60, or 90 min at 37 °C, and was
therefore considered "maximum binding." B,
immunolocalization of p65 following treatment with Grx2 and DA.
Cerebellar granule neurons were exposed to Grx2 or to 600 µM DA for 2 h, fixed, and analyzed by indirect
immunofluorescence using polyclonal anti-p65 antibodies. Note that
NF-
B levels in condensed and apoptotic nuclei are very low.
Bar indicates 10 µm. C, Grx2 increases
I-
B
phosphorylation and degradation. Cerebellar granule neurons
were exposed to Grx2 for different time points as indicated. Cells were
harvested and total proteins isolated from Grx2-treated and control
untreated cells were separated on 12.5% polyacrylamide gel and blotted
onto polyvinylidene difluoride membrane. The membrane was stained with
Ponceau to verify that equal amounts of protein were transferred, after
which the blot was reacted with anti-I-
B
and anti-phosphorylated
I-
B
antibodies (1:1000, New England Biolabs, Beverly, MA).
Secondary antibody was conjugated goat anti-rabbit horseradish
peroxidase. The blots were developed using the ECL system, and a
representative Western blot is shown (n = 4).
B (Fig. 6). Administration of Grx2 (8.3 µM) to
neuronal cells caused a 10-fold stimulation (10.6 ± 2.6) in
NF-
B binding activity (Fig. 6A). In comparison, treatment
of isolated nuclei with Grx2 resulted in a maximum 2-fold stimulation
(Fig. 6A, Nuc + Grx2). The differences in the
extent of activation of NF-
B between whole cells and isolated nuclei
suggested that Grx2 was having an additional cytoplasmic effect. This
effect repeated itself upon DA treatment. Exposure of neuronal cells to
DA reduced the DNA-bound NF-
B by 25 ± 2.2% (Fig.
6A). When Grx2 was administered to isolated nuclear extracts
from DA-treated cells, a maximum 2-fold increase in NF-
B binding was
measured (Fig. 6A, DA + Nuc + Grx2). On the other
hand, administration of DA and Grx2 to whole neurons resulted in 4-fold
increase in NF-
B binding activity (3.75 ± 0.78) (Fig.
6A). Confocal microscopy clearly revealed that Grx2 caused
nuclear accumulation of p65 whereas DA reduced nuclear levels of p65
(Fig. 6B). To further analyze the mechanism by which Grx2
affected NF-
B binding activity, we examined whether Grx2 was
affecting the levels of I-
B
. As shown in Fig. 6C,
administration of Grx2 to neurons led to a time-dependent
increase in I-
B
phosphorylation, which was followed by I-
B
degradation. In conclusion, these results show that the major mechanism
for NF-
B activation following Grx2 treatment is NF-
B
translocation from the cytosol to the nucleus. In addition, nuclear
Grx2 could affect the binding of nuclear NF-
B directly or
indirectly, presumably by reducing the NF-
B proteins.
B Activation Is Essential for Neuronal Viability (Figs.
7 and
8)--
To explore whether NF-
B
activation was important for neuronal survival, neurons were exposed to
SN50, a peptide that inhibits NF-
B translocation (59). Even at low
concentrations (9 µM), SN50 was very toxic (data not
shown), suggesting that inhibition of NF-
B translocation rendered
neurons more vulnerable. However, the peptide could have had other
nonspecific effects and was thus be toxic to neurons through other
mechanisms. To specifically inhibit NF-
B translocation, neurons were
cotransfected with different amounts of I-
B dominant negative gene
(60) (I-
B-
N) and green fluorescent protein (GFP). I-
B-
N
cannot be phosphorylated and thus will not release NF-
B proteins.
Around 0.1% of neurons were cotransfected. To assess whether
overexpression of I-
B-
N inhibited NF-
B translocation in
GFP-positive neurons, cells were cultured on coverslips, fixed, and
reacted with p65 antibodies. Confocal microscopy revealed that the
immunoreactivity of nuclear and cytoplasmic p65 levels was reduced in
the cotransfected neurons (green cell in Fig. 7),
confirming that I-
B-
N inhibited the translocation of NF-
B to
the nucleus.
View larger version (50K):
[in a new window]
Fig. 7.
Overexpression of
I- B-
N reduced p65
immunoreactivity. Cerebellar granule neurons were cotransfected
with I-
B-
N and GFP, fixed, and reacted with anti-p65 antibodies.
A shows fluorescently labeled neuron (GFP-positive cell),
and B demonstrates reduction in p65 immunoreactivity of this
cell (white arrow) compared with nontransfected
surrounding cells. Transfected cells with GFP alone did not show
reduced immunoreactivity of p65 (data not shown). Bar
indicates 10 µm.
View larger version (19K):
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Fig. 8.
Overexpression of
I- B-
N caused neuronal
death. Cerebellar granule neurons were cotransfected with
I-
B-
N and GFP for 24 h, after which the amount of
fluorescently-labeled neurons was monitored as a function of time in
the presence or absence of DA (A). Treating the
cotransfected neurons with Grx2 did not affect their viability
(B). ***, p < 0.001; **, p < 0.025; *, p < 0.05; ··, p < 0.025 between Grx2+DA-treated cells and Grx2+DA-cotransfected cells.
Error bars represent ± S.D. Statistical
analyses were performed with two-tailed Student's t
test.
B translocation affected
neuronal viability. Twenty-four hours after transfection, the number of
cotransfected green fluorescent neurons was identical to control
GFP-transfected cells. Thereafter, the medium was replaced and neuronal
viability was determined (Fig. 8). In the presence of 1 µg/0.2 ml
I-
B-
N, neuronal viability was decreased as a function of time and
reached a steady level of 65 ± 3.3% S.D. after 5 h.
Increasing I-
B-
N to 5 µg/0.2 ml resulted in a similar rate of
neuronal death (59 ± 8.5% S.D.) (Fig. 8A).
Cotransfection of the neurons with pRC/CMV plasmid and GFP had no
effect on neuronal viability, suggesting that cotransfection per
se did not alter the viability. Administration of 600 µM for 5 h resulted in 54% neuronal death.
Cotransfected neurons were slightly but not significantly more
sensitive to DA (52.6% and 45% neuronal death with 1 and 5 µg of
I-
B-
N, respectively) (Fig. 8A). These results are in agreement with our previous experiments (EMSA of NF-
B and
immunolocalization of p65) showing reduced NF-
B activity and
accumulation of p65 in the cytoplasm after DA treatment. Moreover, Grx2
was incapable of rescuing cotransfected
neurons (Fig. 8B).
View larger version (48K):
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Fig. 9.
Ref-1 expression after treatment with Grx2
and/or DA. Cerebellar granule neurons were treated for 2 h
with 8.3 µM Grx2, 600 µM DA and cotreated
with Grx2 and DA. The neurons were fixed, reacted with anti-Ref-1
antibodies and Cy2-conjugated goat anti-rabbit antibodies, and analyzed
by confocal microscopy. The left panels show
immunoreactivity of anti-Ref-1 antibody, the middle
panels show nuclear staining with DAPI, and the
right panels show superposition of DAPI and
anti-Ref-1 immunoreactivity. Bar indicates 10 µm.
B Binding
Activity through Ref-1 (Figs.
9-11)--
In
untreated cells, Ref-1 was preferentially expressed in the nucleus
(Fig. 9, top panel). Two hours of incubation with
DA caused slight nuclear condensation and a dramatic reduction in Ref-1
immunoreactivity (Fig. 9, second panel) (in
accordance with Western blot analysis, not shown). Grx2 significantly
stimulated the expression of both nuclear and cytoplasmic Ref-1 (Fig.
9, third panel). Treating the neurons with Grx2
mutant C9S-C12S did not alter Ref-1 expression (data not shown),
suggesting that the latter is dependent on a redox-active glutaredoxin.
Grx2 could offset the suppression of Ref-1 expression by DA. In the
presence of both DA and Grx2, Ref-1 levels were even higher than those of untreated control neurons (Fig. 9, bottom
panel).
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Fig. 10.
Reduction in Ref-1 expression after
treatment with Ref-1 antisense. A, cerebellar granule
neurons exposed for 2 h to 8.3 µM Grx2 in the
presence of Ref-1 antisense (5 or 10 µM) or Ref-1 sense
(5 or 10 µM) and were reacted with anti-Ref-1 antibodies.
Green staining indicates the presence of antisense/sense
oligonucleotides. Left panel shows Ref-1
immunoreactivity and antisense/sense staining, whereas the
right panel shows only the Ref-1
immunoreactivity. Ref-1 immunoreactivity in untreated cells and in
cells treated with Grx2 is shown in lowest right
and left panel, respectively. Bar
represents 10 µm. B, Western blot analysis of Ref-1 levels
following treatment with Grx2 and antisense (AS, 5 and 10 µM) or sense (S, 5 and 10 µM).
C, quantitative analysis of Ref-1 alterations is represented
as percentage of untreated cells. **, p < 0.025;
··, p < 0.025; ·, p < 0.05 between Grx2-treated neurons and Grx2-treated neurons that were exposed
to Ref-1 antisense. Error bars represent ± S.D. Statistical analyses were performed with two-tailed Student's
t test (n = 5).
View larger version (33K):
[in a new window]
Fig. 11.
NF- B binding
activity in cerebellar granule neurons following treatment with Ref-1
antisense. A, EMSA showing NF-
B DNA binding activity
in nuclear extracts isolated from cerebellar granule neurons treated
with 8.3 µM Grx2 or pre-exposed to 5 and 10 µM antisense and 10 µM sense and then
treated with Grx2. The first lane shows NF-
B
binding activity from untreated granule neurons. B,
quantitative analysis of NF-
B induction is presented as percentage
of untreated cells (n = 4). **, p < 0.025; *, p < 0.05; ··, p < 0.025 between Grx2-treated neurons and Ref-1 antisense-treated neurons.
Error bars represent ± S.D. Statistical
analyses were performed with two-tailed Student's t
test.
B DNA binding activity were
elevated at the same time, it could be that the induction of Ref-1 was
important for the activation of NF-
B. Exposure of neurons to Ref-1
antisense oligonucleotide reduced the expression levels of Ref-1,
whereas the sense sequence had no effect (Fig. 10A). Similar
results were obtained with Western blot analysis (Fig. 10, B
and C). Grx2 could not activate NF-
B in the presence
of Ref-1 antisense oligonucleotide in a concentration-dependent
manner (Fig. 11). However, Grx2 enhanced the DNA binding activity of
NF-
B in the absence of Ref-1 antisense, or in the presence of the
sense oligonucleotide (Fig. 11). These results demonstrate that Grx2 stimulates the DNA binding activity of NF-
B through Ref-1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B-dependent signal transduction pathway through Ref-1. Grx2 penetrated the neurons whether
or not it was redox-active (Fig. 2). Our results are consistent with
several studies that demonstrated that human Trx and its double thiol
mutant were taken up similarly by 3T3 and MCF7 cells (2, 3). The
mechanism by which Grx2 penetrates into the neurons is not clear. It
does not involve a coated pit-dependent mechanism, perhaps
by fluid phase, for example (61). Grx2 internalization per
se is not sufficient to initiate signal transduction; it must catalyze GSH-disulfide oxidoreduction in its active form. Similarly, Gasdaska et al. (3) have shown that the redox activity of
Trx is essential for its effects on cell proliferation. Our study does
not rule out the possibility that Grx2 exerts its effects through
specific interaction with cell surface receptors.
B Rel family of proteins, which have conserved cysteine
residues in their DNA binding domains (62, 63). Modification of the
cysteines with diamide reduced their activity, whereas treatment with
reducing agents enhanced DNA binding. In this work, redox-active Grx2
increased the DNA binding activity of NF-
B. The effect was observed
in isolated nuclei, suggesting a direct effect on NF-
B
(e.g. the reduction of a mixed disulfide between a conserved
cysteine and GSH). However, Grx2 also stimulates NF-
B translocation,
through a process that involves I-
B
phosphorylation and
degradation. Several reports indicated that accumulation of ROS can
induce I-
B phosphorylation and degradation (64-66). Our results
show that a reductive agent such as Grx2 can induce I-
B
phosphorylation and/or ubiquitin-dependent degradation.
This notion is consistent with the findings that glutathione peroxidase
is involved in I-
B
phosphorylation (64). Furthermore,
ubiquitin-conjugating enzymes have been shown to be redox-regulated by
the ratio between GSH and GSSG (67, 68). Our results are partially
consistent with the recent findings obtained by Hirota et
al. (69), who showed that hGrx reduced the ability of tumor
necrosis factor
to phosphorylate I-
B but on the other hand
enhanced NF-
B activation in HEK293 cells.
B DNA activity through Ref-1
activation (Fig. 11) demonstrates a multiple signaling role for Grx2.
It has been shown that AP-1 transcriptional activity is regulated by a
direct association between Trx and Ref-1 (49). Our data show that Ref-1
expression levels are redox regulated. In the presence of DA which
causes oxidative stress, Ref-1 levels were dramatically decreased (Fig.
10). Furthermore, no induction in Ref-1 levels was observed in the
presence of the double thiol mutant. Ref-1 down-regulation was observed
in certain cases of apoptosis, such as the myeloid leukemia cells line
HL-60 (70) or following ischemia (71, 72). It is known that, after
ischemia and reperfusion, mitochondrial production of superoxide
radicals is increased. Thus, the reduced expression of Ref-1 after
ischemia or DA treatment may be due to oxidative damage.
-hydroxyethyl disulfide and GSH as a substrate (51). However, their
protective profiles against DA-induced apoptosis were almost identical
(Fig. 1). This may simply reflect that the two proteins have similar catalytic properties to their nonidentified in vivo substrate.
B shows that the DNA binding of NF-
B
is regulated by a GSH mixed disulfide mechanism.
B to cell death and survival pathways is an
intriguing and controversial issue. Our results show that NF-
B
activation is important for the viability of cerebellar granule neurons
and that Grx2 exerts its protective effects through the activation of
NF-
B signaling pathways. First, inhibition of NF-
B translocation
and binding by I-
B-
N overexpression causes neuronal death (Fig.
8). Second, Grx2 could not rescue the neurons overexpressing
I-
B-
N (Fig. 8). Third, DA-induced neuronal death is correlated
with a significant reduction in NF-
B DNA binding activity (Fig. 3)
and a decrease in p65 levels in condensed and apoptotic nuclei (Fig.
6B). Our findings are in agreement with several recent
reports suggesting a role for NF-
B in protection from apoptosis.
Abrogation of NF-
B, either by deletion of RelA (p65) or by
overexpression of I-
B-
N, sensitizes immune cells to apoptosis in
response to tumor necrosis factor and DNA damaging agents (33, 34, 75,
76). NF-
B activation protected hippocampal neurons against oxidative
stress-induced apoptosis (77). Furthermore, nerve growth factor
protects sympathetic neurons by the activation of NF-
B (78).
Increased NF-
B activity was found in surviving endothelial cells,
whereas apoptotic cells show caspase-mediated cleavage of p65 (79). It
was further shown that uncleavable caspase-resistant p65 protected the
cells from apoptosis (79). In contrast, NF-
B has been implicated in
the death of neurons induced by glutamate and
-amyloid protein (80,
81), by ceramide-induced apoptosis in mesencephalic dopaminergic
neurons (43), and in leukemia cells (82). It is possible that the
opposing roles of NF-
B in cell survival and death stem from the
diversity in subunit expression and subcellular compartmentation. In
different cell types, the relative expression of the different members
of the NF-
B family may influence the composition of the dimers and, as a consequence, the affinity of the activated complex to specific DNA
sequence motifs located in the regulatory region of different genes.
Moreover, activation of distinct patterns of genes by the simultaneous
activation of other regulatory transcription factors may contribute to
opposite cell fates in different cell types as well as within the same
cell type. Tamatani et al. (83) showed that tumor necrosis
factor protects hippocampal neurons by the induction of
bcl-2 and bcl-x through the activation of
NF-
B. These observations strengthen the notion that NF-
B can
inhibit apoptosis by inducing the expression of antiapoptotic genes.
B
signaling pathways through Ref-1. Inhibition of NF-
B activity by
overexpression of I-
B-
N causes neuronal death, indicating that
the activation of a NF-
B signaling cascade is necessary to granule
neuron survival. Studies will be needed to identify Grx- and
NF-
B-induced genes that promote neuronal survival, and to clarify
the precise role of redox regulation by glutaredoxin in signal
transduction and neuronal protection.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Israeli Academy of Sciences and Humanities and the Israeli Ministry of Health, by Swedish Cancer Society Grant 961, and by Swedish Medical Research Council Grant 03XS-13005-01A (to A. H. and A. V.-G.).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.
** Supported by a Sackler fellowship at Tel Aviv University.
To whom correspondence should be addressed: Dept. of
Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-3-6409782; Fax: 972-3-6407643; E-mail: barzilai@post.tau.ac.il.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008121200
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ABBREVIATIONS |
---|
The abbreviations used are: DA, dopamine; Grx, glutaredoxin; DTT, dithiothreitol; ROS, reactive oxygen species; EMSA, electrophoretic mobility shift assay; DAPI, 4,6-diaminodiphenyl-2-phenylindole; GFP, green fluorescent protein; PBS, phosphate-buffered saline.
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