From the Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, the ¶ Department of Neurology and
Felsenstein Medical Research Institute, Rabin Medical Center, and the
Department of Clinical Biochemistry, Sackler School of Medicine, the
Interdepartmental Core Facility, Sackler School of
Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel, and
the § Department of Biochemistry and Biophysics, Medical
Nobel Institute for Biochemistry, Karolinska Institute,
Stockholm S-171 77 Sweden
Received for publication, February 14, 2001, and in revised form, March 27, 2001
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ABSTRACT |
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Glutaredoxin 2 (Grx2) from Escherichia
coli protects cerebellar neurons from dopamine-induced apoptosis
via nuclear factor kappa B (NF- Excessive production of reactive oxygen species in living
cells may damage their biological components. This condition, referred to as oxidative stress, is a common denominator of pathological conditions. Cells have evolved a wide array of antioxidant mechanisms including small reducing molecules (e.g. glutathione,
ascorbic acid), antioxidative enzymes (e.g. catalase,
superoxide dismutase, glutathione peroxidase; for review, see Refs.
1-4), and oxidoreductase enzymes such as thioredoxin and glutaredoxin
(Grx)1 (5).
Grx are antioxidant enzymes by virtue of the reducing power of their
active site (CXXC), which catalyzes the transfer of
electrons from reduced glutathione to disulfides (5). This thiol
disulfide interchange reaction is crucial for the maintenance of
intracellular redox homeostasis, especially under oxidative stress (6).
Mammalian Grx is widely expressed in different cell types, including
neurons (7-11). The enzyme can restore the activity of
glutathionylated proteins containing mixed disulfides between a protein
thiol and GSH (inactive form) by reducing the disulfide bridge to give
reduced GSH and the active protein form containing a free thiol.
Examples of such GSH-thiol regulation of activity can be found in
tyrosine phosphatase 1B (13), phosphofructokinase (14, 15), nuclear factor-I (16), and polyomavirus enhancer-binding protein 2 (17). Thanks
to the antioxidant properties of Grx activity, human Grx and
Escherichia coli Grx2 can rescue cerebellar granule neurons from dopamine (DA)-induced oxidative stress (18).
DA, the endogenous neurotransmitter of the nigrostriatal pathway, is a
powerful oxidant that exerts its toxic effects through its oxidative
metabolites. DA-induced oxidations are generally implicated in
neurodegenerative processes (19, 20) especially in Parkinson's disease
(21, 22). Administration of DA to rat striatum caused pre- and
postsynaptic damage (23). Intraventricular injection of DA in rats
resulted in dose-dependent death of the animals (24).
In vitro studies have shown that DA can cause cell death in
mesencephalic, striatal, and cortical primary neuron cultures (25-29).
DA-induced cell death in sympathetic, cerebellar granule neurons, PC-12
cells, and thymocytes has all the features of apoptotic cell death
(30-32). Apart from the administration of Grx, the toxic effects of DA
can be prevented by the application of small molecular weight
antioxidants such as N-acetylcysteine, catalase, ascorbic
acid, and dithiothreitol (30, 31, 33, 34).
Little is known about the molecules and signaling pathways involved in
DA-induced apoptosis. Enhancement of the DNA binding activity of
NF- Ras is a family of proteins involved in the regulation of cell
proliferation, cytoskeletal rearrangements, and differentiation and
survival of different cell types (37, 38). Ras activation is localized
on the inner surface of the cell membrane where it cycles between two
states: a GDP-bound inactive state and a GTP-bound active state. This
cycle is regulated by cell surface receptors including tyrosine kinase
receptors. Ras-GTP was shown to interact and activate PI3K, a
heterodimer composed of an 85-kDa regulatory subunit and a 110-kDa
catalytic subunit. PI3K activity is generally regulated by tyrosine
kinase receptors (39). Upon stimulation, PI3K phosphorylates
phosphatidylinositol molecules and generates the phosphorylated
products phosphatidylinositol (3,4,5)-trisphosphate and
phosphatidylinositol (3,4)-bisphosphate, which bind to the pleckstrin
homology domain of the serine/threonine kinase Akt (also known
as protein kinase B). Akt is then translocated to the plasma membrane
where it undergoes phosphorylation by PDK1 and 2 (40-42). Growth
factors and cytokines can also activate Akt via the PI3K pathway (43).
Activated Akt can phosphorylate and inactivate proapoptotic proteins
such as Bad, procaspase 9, glycogen synthase kinase-3, and members of
the Forkhead transcription factor family. Alternatively, Akt can
activate antiapoptotic proteins such as NF- NF- The AP-1 superfamily consists of several subfamilies including Jun, Fos
and ATF-2, all of which possess a leucine zipper domain that assists
their dimerization (64). The AP-1 proteins form homo- and heterodimers
before binding to their DNA target sites. The activation of AP-1 is
regulated by JNK, extracellular signal-regulated kinase, and p38
mitogen-activated protein kinases (65, 66). Experiments with
sympathetic and cerebellar granule neurons, as well as with PC-12
cells, revealed that JNK/c-Jun signaling promotes apoptosis after
withdrawal of survival factor (67-69). Studies using JNK ( Redox factor-1 (Ref-1) is a multifunctional protein that stimulates the
DNA binding of numerous transcription factors, among them Fos, Jun, and
NF- Our results show that Grx2 protects neurons against DA toxicity through
the activation of both Ras/PI3K/Akt and JNK/AP-1 pathways, which
culminate in 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 was from
Amersham Pharmacia Biotech. Rabbit antibodies against NF- Primary Culture of Cerebellar Granule Neurons--
Cultures of
highly enriched granule neurons were obtained from postnatal day 8 BALB/c mice (80). Cells were dissociated by trypsinization and plated
in standard medium basal medium Eagle's, 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 (81) on
dishes coated with poly-L-lysine (cell density, 7 × 106 cells/35-mm-diameter dish, 2.5 × 105
cells/well for a 96-well plate). 10 µM cytosine
Treatments--
DA (3-hydroxytyramine hydrochloride; 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 and/or Grx2 for various periods of time. Control cultures were maintained in
serum-free standard medium. Prior to addition to the neurons, Grx2 was
reduced with 2 mM dithiothreitol (DTT) for 20 min at 37 °C. DTT was removed by two spin columns of Sephadex G-25
equilibrated with phosphate-buffered saline (PBS). Wortmannin and FTS
were incubated for 1 h in serum-free standard medium before
administration of DA and Grx2.
Analysis of Neuronal Viability--
The viability of the
cultures (96-well plates) was assessed by Alamar Blue assay (AccuMed).
Alamar reagent (1:10) was added to the treated cells for 2 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.
Protein Contents--
Protein cell content was determined
according to the method of Bradford (83) 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 (Sigma). Treated
neurons were fixed for 30 min in ice-cold 4% paraformaldehyde and PBS, washed with PBS (pH 7.4), permeabilized for 15 min with 0.2% Triton X-100, and blocked with 14 µg/ml normal goat IgG and 1% bovine serum
albumin for 1 h. Cells were then incubated with anti-p65 or
anti-Ref1 antibodies (1:50) in PBS containing 1% bovine serum albumin
for 1 h at 37 °C, washed, and incubated for 1 h with
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 5 µg/ml DAPI for 5 min. Nuclei were
visualized under UV light.
Confocal Microscopy--
Fluorescently stained cells were
analyzed using Zeiss confocal laser scanning microscope (CLSM). Zeiss
LSM 410 inverted (Oberkochen, Germany) is equipped with a 25-mW
krypton-argon laser (488 and 568 maximum lines) and 10-mW He-Ne laser
(633 maximum lines). A 40 × NA/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 (84), using 12.5% polyacrylamide gel
electrophoresis. Each lane was loaded with an equal amount of protein
extracts (50 or 100 µg) which, after 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-Ref-1, anti-phospho-JNK, anti-JNK, anti-phospho-Akt, anti-Akt (1:1,000), and Pan anti-Ras (1:2,000) antibodies. For the detection of phospho-JNK,
EnVisionTM+/horseradish peroxidase (rabbit, 1:100) (DAKO, Glostrup,
Denmark) was used to enhance sensitivity. Intensity of the signal was
determined by ECL-Plus detection system (Amersham Pharmacia
Biotech).
Nuclear and Cytoplasmic Extracts--
Neurons (7 × 106 cells) were washed with PBS, 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 assay (EMSA).
EMSA--
The binding reaction mixture containing 10 mM Tris-HCl, pH 7.9, 60 mM KCl, 0.4 mM DTT, 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 and JNK1/2
antisense oligonucleotides and the complementary sense oligonucleotides
were synthesized and high performance liquid chromatography (HPLC)
purified by Sigma Genosys Ltd., Israel. The oligonucleotides were
phosphorotioated at the 3'-end (3 last bases) to confer
nuclease resistance. The sequence of the Ref-1 antisense probe was
5'-TTCCCCGCTTTGGCATCGC-3' and the sense 5'-GCGATCCCAAAGCGGGGAA-3' (18).
The sequence of the JNK1/2 antisense probe was
5'-CGGTAGGCTCGCTTAGCATG-3' and the sense 5'-TGAGGCGTTAAGACGTTCAA-3', as
described by Shan et al. (85). Cerebellar granule neurons
were treated with oligonucleotides 6 days after plating. The JNK1/2
antisense was given 2 h before treatment with Grx2 and/or DA.
PI3K Activity--
Cerebellar granule neurons were washed with
PBS and lysed in lysis buffer containing 20 mM Tris-HCl, pH
7.6, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.2 mM NaVO4,
1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and
10% glycerol. Cleared lysates were normalized for protein content, and
150 µg of protein was rotated with 2 µl of anti-p85 for 2 h at
4 °C. Thereafter protein G-Sepharose beads were added for overnight
incubation. The immunoprecipitates were washed twice with 10% Nonidet
P-40 in PBS and 0.2 mM NaVO4, once with 0.5 M LiCl, 100 mM Tris-HCl, pH 7.6, 0.2 mM NaVO4, and once with TNE buffer (10 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM EDTA, 0.2 mM NaVO4). Kinase
reaction was carried out for 15 min at room temperature in kinase
buffer consisting of 5 mM HEPES pH 7.5, 25 mM
MgCl2, phosphatidylinositol (100 µg/ml sonicated in 10 mM HEPES, 1 mM EGTA), 250 µM ATP,
and 5 µCi of [32P]ATP. The reaction was stopped with 80 µl of 1 N HCl and phospholipids extracted once with 160 µl CHCl3:MeOH (1:1), once with 100 µl of 1 N HCl:MeOH (1:1), and the organic phase was dried under
N2 and resuspended in 10 µl of CHCl3:MeOH
(1:1). Phosphorylated products were resolved on oxalate-impregnated
Silica 60 plates (Merck) using CHCl3, MeOH, and 4 M NH4OH (9:7:2) as solvent. Radioactive products were visualized and quantitated by PhosphorImaging.
Ras Activity Assay--
Activation of Ras was determined by
immunoprecipitation of Ras with GST-RBD (86), precoupled to
glutathione-agarose beads prior to incubation with cell lysates. Cells
were treated with FTS or DA (with or without Grx2) for different times,
washed with PBS, and lysed in lysis buffer containing 50 mM
Tris-HCl, pH 7.6, 150 mM NaCl, 20 mM
MgCl2, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 0.1 µg/ml
leupeptin, 1 mM benzamidin, and 1 mM DTT. The
lysates were spun for 15 min at 14,000 rpm at 4 °C. Precoupled
GST-RBD was added to 100-150 µg of supernatant proteins and
incubated for 30 min at 4 °C. The beads were collected and rinsed
three times in lysis buffer, and bound Ras-GTP proteins were eluted
with sample buffer. Samples were analyzed by Western immunoblotting
with Pan-Ras as described above.
Grx2 Protection Is Abolished by Ras and PI3K Inhibitors--
To
determine whether the protective effect of Grx2 against DA-induced
apoptosis was through the activation of the Ras/PI3K signaling pathway,
cerebellar granule neurons were exposed to 600 µM DA with
or without Grx2 for 5 h in the presence or absence of the Ras
inhibitor FTS or PI3K inhibitor wortmannin. Both Ras and PI3K
inhibitors blocked the protective effect of Grx2 on DA-treated cells
(Fig. 1, 27 ± 8% and 33 ± 6%, respectively). Wortmannin had no apparent effect on the viability
of untreated neurons, whereas inhibition of the
Ras-dependent pathways by FTS caused a significant
reduction in neuronal viability (58 ± 4%). These results suggest
that Grx2 protects neurons through the Ras/PI 3-kinase pathway.
Inhibition of the Ras/PI3K Pathway Prevents the Activation of
NF- Grx2 Stimulates Ras and PI3K, but DA Inhibits PI3K
Activity--
We next examined the effect of Grx2 and DA on active Ras
and active PI3K forms (Figs. 3 and
4, respectively). These activities were
assayed before the commitment point for DA-induced apoptosis, which was
shown previously to be 2 h (18). Both active Ras and active PI3K
forms were elevated significantly after exposure of the cells to Grx2
for 1 and 2 h (162 ± 36% and 170 ± 26%, respectively (Ras activation), 124 ± 4.9% and 132 ± 6.8%, respectively
(PI3K activation)). The effect of Grx2 was not observed before 1 h
had elapsed (data not shown). DA had no significant effect on the active Ras form but inhibited PI3K activity significantly (77 ± 7.8% and 35 ± 2% after 1- and 2-h DA exposure, respectively). FTS alone down-regulated active Ras as expected, also causing a small
but significant decrease in PI3K activity (88 ± 1.6%, 2-h FTS).
In the presence of FTS, Grx2 could not restore active Ras or active
PI3K (30 ± 11% and 83 ± 9.2; 56 ± 26% and 79 ± 4% for 1 and 2 h, respectively). FTS and wortmannin attenuated
the ability of Grx2 to confer neuronal protection against DA toxicity (Fig. 1). In addition, both compounds inhibited the activation of the
Ras/PI3K pathway by Grx2 (Fig. 2). Therefore, the Ras/PI3K pathway
could be a mechanism by which Grx2 exerts its protective effect on
the neurons.
Grx2 Up-regulates the Activity of Akt, but DA Down-regulates
It--
PI3K can activate Akt, thus promoting the survival of
cerebellar granule neurons (49). Furthermore, Akt was shown to promote cell survival via NF- Grx2 Regulates Akt Activity through Ref-1--
It was shown that
Grx2-induced activation of NF- Grx2 Up-regulates the DNA Binding Activity of AP-1 via Ref-1, but
DA Down-regulates It--
Ref-1 can stimulate the DNA binding activity
of AP-1, which is known to be involved in cell survival and apoptosis.
Exposure of cerebellar granule neurons to Grx2 augmented the DNA
binding activity of AP-1 significantly (Fig.
8) as early as 1 h after exposure
(240 ± 20%) and returned to basal levels thereafter. In
contrast, DA significantly decreased the binding activity of AP-1 in a
time-dependent manner (73 ± 5%) (Fig. 8). The DNA
binding activity of AP-1 was inhibited by wortmannin or Ref-1 antisense oligonucleotide. Similarly, wortmannin could also inhibit human Grx
ability to activate AP-1 binding (data not shown). Similar results were
obtained using human Grx (data not shown). These results suggest that
Grx2 affected the activation of AP-1 via PI3K and/or Ref-1.
Grx2 Activates JNK, but DA Down-regulates It--
Levels of
phosphorylated JNK were elevated significantly as early as 10 min
(134 ± 2%) after exposure to Grx2 (Fig.
9); they peaked at 1 h (228 ± 45%) and decreased 1 h later. Phosphorylation of JNK was
down-regulated by treatment with DA (67 ± 7.5% and 53 ± 4.3% for 1 and 2 h, respectively) (Fig. 9). These results are in
accordance with the activation profile of AP-1 (Fig. 8). Wortmannin had
no significant effect on the phosphorylation levels of JNK (data not
shown).
Grx2 Activates NF- Intracellular redox status has been linked to cellular
differentiation, immune response, growth control, tumor progression, and apoptosis (88). Oxidants and antioxidants can act as signaling molecules that modify the function of enzymes such as phosphatases and
kinases and directly or indirectly affect the activity of many
transcription factors (e.g. c-Jun, c-Fos, Myb, p53) or
nuclear receptors such as the glucocorticoid and estrogen receptors
((89) for review, see Refs. 90 and 91). Additional research is needed to clarify the role of redox regulation in the survival of neurons and
in neurodegenerative diseases.
Human Grx and E. coli Grx2 can protect neurons from
DA-induced apoptosis (18). The effect is specifically related to Grx activity and does not exist in a general reductant molecule. Whereas GSH confers neuronal protection by scavenging DA oxidative metabolites, Grx acts by a different mechanism because it is not a free radical scavenger (data not shown). In addition, GSH at the mM
range had a much less protective effect and showed a lower capability
of activating NF- Our work suggests that Grx2 exerts its protective effects through
activation of two separate signaling pathways: the Ras/PI3K/Akt and
JNK/AP-1 pathways, which culminate in the stimulation of NF- The mechanism by which Grx2 stimulates Ras and JNK activities is not
known. It has been suggested that changes in the redox state may alter
Ras and JNK activities directly. Because both enzymes have a critical
cysteine residue in their active site, they could be affected by the
redox environment (35, 92). For example, in oxidizing conditions, the
thiol of the active site could form a mixed disulfide with GSH, leading
to an inactive protein-S-SG species. Grx2 may reduce this mixed
disulfide, producing non-glutathionylated and active proteins. This is
precisely how Grx2 and other E. coli Grxs activate
glutathionylated arsenate reductase (93). Whereas the 1-h delay in the
activation of Ras by Grx2 argues against direct activation of Ras,
early JNK activation by Grx2 supports the notion that Grx2 directly
activates JNK. Further studies are needed to determine whether the
activity of Akt could also be regulated by a mixed disulfide mechanism.
JNK has been implicated in cell death and survival (70 and references
therein). We suggest an anti-apoptotic role for the JNK/AP-1 pathway
which is in agreement with recent works (70, 94-97) but in
disagreement with other studies performed on cerebellar granule neurons
and experiments in which DA-induced apoptosis was mediated by AP-1
activation (52, 68, 69, 98-102). It may be that activation of
JNK/c-Jun after inhibition of PI3K (52, 104) is an attempt to activate
rescue processes for the prevention of cell death. The apparent dual
role of the JNK pathway may reflect the activation of specific Fos/Jun
components of AP-1 leading to the transcription of different genes
and/or interaction with opposing regulatory signaling pathways.
Our results indicate cross-talk between the Ras/PI3K/Akt and JNK/AP-1
pathways: inhibition of PI3K abolished the ability of Grx2 to stimulate
AP-1 binding activity, whereas inhibition of JNK prevented the
activation of NF- Ref-1 affected both the AP-1 and NF- In summary (Fig. 12), we demonstrate
that Grx2 exerts its protection against DA-induced apoptosis through
activation of the Ras/PI3K/Akt and JNK/AP-1 pathways, culminating in
NF-B) activation, which is mediated by
the expression of redox factor-1 (Ref-1). An analysis of the mechanisms
underlying Grx2 protective activity revealed dual activation of signal
transduction pathways. Grx2 significantly activated the
Ras/phosphoinositide 3-kinase/Akt/NF-
B cascade in parallel to
the Jun N-terminal kinase (JNK)/AP1 cascade. Dopamine, in
comparison, down-regulated both pathways. Treatment of neurons with
Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate
Akt and AP-1 but had no effect on the phosphorylation of JNK1/2,
suggesting that Akt/NF-
B and AP-1 are regulated by Ref-1. Exposure
of the neurons to JNK1/2 antisense oligonucleotide in the presence of
Grx2 significantly reduced AP-1 and NF-
B DNA binding activities and
abolished Grx2 protection. These results demonstrate that dual
activation of Ras/phosphoinositide 3-kinase and AP-1 cascades, which
are mediated by Ref-1, is an essential component of the Grx2 mechanism
of action.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B protects neurons from DA-induced apoptosis (18). A survival
signal pathway that might activate NF-
B is the
Ras/PI3K/Akt/NF-
B cascade (35, 36).
B (44-47). Akt is
required for nerve growth factor-induced survival in sympathetic
neurons (48) and for PI3K-induced survival of cerebellar granule
neurons (49-53).
B and AP-1 are transcription factors that are critically
important for cell survival and apoptosis. NF-
B consists of homo- or
heterodimers of p50, p52, p65 (RelA), RelB or c-Rel. NF-
B proteins
are usually expressed in an inactive form, bound to proteins inhibiting
their activity named I-
B. Following the appropriate cellular
stimulation, I-
B become phosphorylated by the multisubunit I-
B
kinase complex, which subsequently targets I-
B for ubiquitination
and degradation by the proteosome (54). The free NF-
B dimer
translocates into the nucleus and up-regulates transcription of
specific genes (55-57). The DNA binding activity of NF-
B is
activated in brain injury models (brain trauma, focal ischemia,
kainate-induced seizure) (58-61) and in the brains of Alzheimer's and
Parkinson's disease patients (62, 63).
/
) mice
suggested that JNK promotes apoptosis in the hippocampus and in the
developing embryonic neural tube. However, in the embryonic forebrain,
JNK proteins have the opposite function and are necessary for the
survival of developing cortical neurons (70). The JNK pathway is also
activated in some experimental models of Parkinson's disease (71,
72).
B through redox regulation (73). Ref-1 possesses
apurinic/apyrimidinic endonuclease DNA repair activity against DNA
damage caused by reactive oxygen species, UV and infrared radiation
(73-75) and acts as a repressor of genes encoding calcium-responsive elements (76). It is expressed in subpopulations of cells in the brain,
including cerebellar granule neurons (77, 78).
B activation. In contrast, DA down-regulated these
two pathways.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B p65 and
Ref-1 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,
CA); rabbit anti-PI3K p85 was purchased from Upstate Biotechnology
(Lake Placid, NY). Polyclonal antibodies against phospho-JNK/stress-activated protein kinase, JNK, phospho-Akt (Ser-473), and Akt were from New England Biolabs (Beverly, MA). Monoclonal Pan-Ras was from Oncogene (Cambridge MA). E. coli
Grx2 was a recombinant preparation purified to homogeneity as described (79). Phosphatidylinositol and wortmannin were from Sigma.
S-trans, trans-farnesyl thiosalicylic acid (FTS)
was kindly provided by Prof. Kloog, Department of Neurobiochemistry,
Tel Aviv University.
-D-arabinofuramoside (Ara-C) was added to the medium
18-22 h after plating to prevent replication of non-neuronal cells
(82).
B or AP-1
oligonucleotides (5'-AGTTGAGGGGACTTTCCCAGGC-3' and
5'-CGCTTGATGAGTCAGCCGGAA-3', respectively) (Promega, Madison WI) was
incubated for 30 min with 5 µg of nuclear extract. For AP-1 binding
activity, the reaction was done on ice. For specificity control, a
50-fold excess of unlabeled probe was applied. 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 PhosphorImaging (Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The neuroprotective activity of Grx2 is
abolished by Ras and PI3K inhibitors. Cerebellar granule neurons
were incubated with 600 µM DA, 100 nM
wortmannin (W), or 25 µM FTS in the presence or absence
of 20 µM Grx2. After 5 h of incubation, cell
viability was determined by Alamar Blue assay (n = 4).
***, p < 0.001; **, p < 0.025. Error bars represent ± S.D. Statistical analyses were
performed with a two-tailed Student's t test. C,
control.
B by Grx2--
We have shown previously that activation of the
DNA binding activity of NF-
B is essential to the viability of
cerebellar granule neurons exposed to DA (18). To examine whether the
inhibition of Ras and PI3K affected Grx2-dependent NF-
B
activation, neurons were exposed to Grx2 for 2 h in the presence
or absence of FTS or wortmannin and the NF-
B DNA binding activity
analyzed (Fig. 2, A and
B). Administration of Grx2 to neuronal cells caused a 10-fold stimulation in NF-
B binding activity (Fig. 2, A
and B) and the nuclear accumulation of p65 (Fig.
2C). However, NF-
B activation was completely abolished by
FTS and wortmannin (215 ± 180% and 173 ± 53%,
respectively) (Fig. 2, A and B). Moreover, wortmannin reduced both cytoplasmic and nuclear levels of p65 and
caused nuclear condensation, which is a typical feature of apoptotic
nuclei (Fig. 2C). Similarly, treating the neurons with human
Grx resulted in NF-
B stimulation, which was abolished by FTS and
wortmannin (data not shown). These results suggest that Grx2 and human
Grx activated NF-
B through the Ras/PI3K pathway.
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Fig. 2.
Inhibition of the Ras/PI3K pathway prevents
activation of NF- B by Grx2. Panel
A, EMSA showing the DNA binding activity of NF-
B in nuclear
extracts from cerebellar granule neurons incubated with 20 µM Grx2 in the presence/absence of 100 nM
wortmannin (W) or 25 µM FTS for 2 h. Identical
cultures were incubated in serum-free medium and served as controls.
Panel B, quantitative analysis of NF-
B induction is
represented as a percent of untreated cells (n = 4).
***, p < 0.001; ···,
p < 0.001 versus Grx2 activation.
Error bars represent ± S.D. Statistical analyses were
performed with a two-tailed Student's t test. Panel
C, immunolocalization of p65 after treatment with Grx2 and
wortmannin. Cerebellar granule neurons were exposed to Grx2 alone or to
Grx2 and wortmannin for 2 h, fixed, reacted with anti-p65
antibodies and Cy2-conjugated goat anti-rabbit antibodies, and analyzed
by confocal microscopy. The left frames show
immunoreactivity of anti-p65 antibody, the middle frames
show nuclear staining with DAPI, and the right frames show
superposition of DAPI and anti-p65 immunoreactivity. Note the
appearance of condensed nuclei after wortmannin treatment and their low
level of p65 protein. Bar = 10 µm.
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Fig. 3.
Alterations in Ras activity after treatment
with Grx2/DA. Panel A, cerebellar granule neurons were
treated with 20 µM Grx2, 600 µM DA, 25 µM FTS, or 20 µM Grx2 and 25 µM FTS for 1 and 2 h and assayed for Ras activity as
described under "Materials and Methods." Panel B,
quantitative analysis of Ras activity is represented as a percent of
untreated cells (n = 4). *, p < 0.05. Error bars represent ± S.D. Statistical analyses were
performed with a two-tailed Student's t test. C,
control.
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Fig. 4.
Alterations in PI3K activity after treatment
with Grx2/DA. Cerebellar granule neurons were treated with 20 µM Grx2, 600 µM DA, 25 µM
FTS, or 20 µM Grx2 and 25 µM FTS for 1 and
2 h and assayed for PI3K activity as described under "Materials
and Methods." Quantitative analysis of PI3K activity is represented
as a percent of untreated cells (n = 4). ***,
p < 0.001; **, p < 0.025; *,
p < 0.05. Error bars represent ± S.D.
Statistical analyses were performed with a two-tailed Student's
t test. C, control.
B activation (87). To investigate whether Akt
is involved in the Grx2 protective effect, we monitored the levels of
phospho-Akt (the active form of Akt) after Grx2 and DA treatments (Fig.
5). Administration of Grx2 significantly
increased the levels of phosphorylated Akt, whereas administration of
DA decreased them (240 ± 35% and 44 ± 8%, respectively).
Grx2-induced phosphorylation was blocked by wortmannin (73 ± 13%), an inhibitor of PI3K. Therefore, the activation of Akt via Grx2
may be dependent on PI3K.
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Fig. 5.
Grx2 up-regulates but DA down-regulates Akt
activity. Panel A, cerebellar granule neurons were
treated with 20 µM Grx2 or 600 µM DA in the
presence or absence of 100 nM wortmannin (W). Total
cellular proteins were extracted and immunoreacted with
anti-phospho-Akt antibody, as described under "Materials and
Methods." Panel B, quantitative analysis of Akt
phosphorylation is represented as a percent of untreated cells
(n = 4). ***, p < 0.001;
···, p < 0.001 versus
Grx2 activation. Error bars represent ± S.D.
Statistical analyses were performed with a two-tailed Student's
t test. No changes in total Akt levels were detected in
these experiments (data not shown). C, control.
B is mediated by up-regulating the
expression of Ref-1. DA, in comparison, down-regulated Ref-1 levels
(18). To determine whether the PI3K pathway was involved in the
activation of Ref-1, we monitored the expression levels of Ref-1 in
neurons treated with Grx2 in the presence or absence of wortmannin
(Fig. 6). Grx2 significantly stimulated
the expression levels of both nuclear and cytoplasmic Ref-1, whereas
wortmannin caused nuclear condensation and a dramatic reduction in
Ref-1 immunoreactivity. Western blot analysis (Fig. 6B)
revealed a significant increase in Ref-1 levels after exposure to Grx2
only (175 ± 40% after 1 h and 404 ± 46% after 2 h). Wortmannin abolished the ability of Grx2 to elevate Ref-1 levels
(105 ± 11 and 31 ± 14% after 1 and 2 h,
respectively). Because Ref-1 and Akt activate NF-
B in a
PI3K-dependent manner, it could be that one may activate
the other. To determine the order of activation, neurons were exposed
to Grx2 in the presence or absence of Ref-1 antisense oligonucleotide
(Fig. 7). This Ref-1 antisense
oligonucleotide was shown to inhibit Ref-1 expression (18). The Ref-1
antisense oligonucleotide reduced the stimulation of Akt by Grx2
significantly (93 ± 27%). The sense sequence had no effect
(228 ± 60%) (Fig. 7). These results demonstrate that Akt is a
downstream target of Ref-1.
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Fig. 6.
Activation of Ref-1 by Grx2 is dependent on
PI3K. Panel A, immunolocalization of Ref-1 after
treatment with Grx2 and wortmannin (W). Cerebellar granule neurons were
exposed to 20 µM Grx2 in the presence or absence of 100 nM wortmannin for 2 h, fixed, and reacted with
anti-Ref-1 and Cy2-conjugated goat anti-rabbit antibodies, and analyzed
by confocal microscopy. The left frames show the
immunoreactivity of anti-Ref-1 antibody, the middle frames
show nuclear staining with DAPI, and the right frames show
superposition of DAPI and anti-Ref-1 immunoreactivity. Note that the
condensed nuclei after wortmannin treatment express low levels of
Ref-1. The bar indicates 10 µM. Panel
B, Western blot analysis of Ref-1 levels after exposure of
cerebellar granule neurons to 20 µM Grx2 in the presence
or absence of 100 nM wortmannin for 1 and 2 h.
Panel C, quantitative analysis of Ref-1 levels is
represented as a percent of untreated cells (n = 4).
***, p < 0.001; **, p < 0.025; *,
p < 0.05; ·, p < 0.05 versus Grx2 activation. Error bars represent ± S.D. Statistical analyses were performed with a two-tailed
Student's t test
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Fig. 7.
Ref-1 antisense blocks Grx2-induced Akt
activation. Upper panel, cerebellar granule neurons
were treated with 20 µM Grx2 in the presence of Ref-1
antisense (AS) or sense (S) oligonucleotides (5 µM).
Total cellular proteins were extracted and immunoreacted with
anti-phospho-Akt antibody, as described under "Materials and
Methods." Lower panel, quantitative analysis of Akt
phosphorylation is represented as a percent of untreated cells
(n = 3). ***, p < 0.001;
··, < 0.025 versus Grx2 activation.
Error bars represent ± S.D. Statistical analyses were
performed with a two-tailed Student's t test.
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Fig. 8.
AP-1 binding activity in cerebellar granule
neurons after treatment with Grx2, DA, wortmannin, and Ref-1 sense and
antisense oligonucleotides. Panel A, EMSA showing AP-1
DNA binding activity in nuclear extracts from cerebellar granule
neurons incubated with 20 µM Grx2 in the presence or
absence of 100 nM wortmannin (W) or Ref-1 antisense (AS)
and sense (S) oligonucleotides (5 µM), and with 600 µM DA for 1 and 2 h. Identical cultures were
incubated in serum-free medium and served as control (C). Panel
B, quantitative analysis of AP-1 induction is represented as a
percent of untreated cells (n = 4). ***,
p < 0.001; ···, < 0.001; and
··, p < 0.025 versus Grx2
activation. Error bars represent ± S.D. Statistical
analyses were performed with a two-tailed Student's t
test.
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Fig. 9.
Grx2 elevates, whereas DA down-regulates,
levels of phosphorylated JNK. Panel A, cerebellar
granule neurons were treated with 20 µM Grx2 or 600 µM DA for different time points, after which total
cellular proteins were extracted and immunoreacted with
anti-phospho-JNK antibody, as described under "Materials and
Methods." Panel B, quantitative analysis of JNK
phosphorylation is represented as a percent of untreated cells
(n = 3). ***, p < 0.001; *,
p < 0.05. Error bars represent ± S.D.
Statistical analyses were performed with a two-tailed Student's
t test. No changes in total JNK levels were detected (data
not shown). C, control.
B through the JNK/AP-1 Signaling
Pathway--
To evaluate the importance of the JNK/AP-1 pathway in the
Grx2 protective mechanism, neurons were exposed to a JNK1/2 antisense oligonucleotide (85) prior to treatment with Grx2. This treatment reduced the expression levels of phospho-JNK (77 ± 32%) and the DNA binding activity of its downstream effector AP-1 (65 ± 45%). The sense sequence had no significant effect. The antisense
oligonucleotide also blocked Grx2-induced NF-
B activation (152 ± 93% compared with 1,100 ± 160% Grx2 alone) (Fig.
10, E and F).
Accordingly, the JNK1/2 antisense oligonucleotide significantly reduced
the ability of Grx2 to confer neuronal protection against DA toxicity (54 ± 9% compared with 98 ± 1%; Fig.
11). Therefore, Grx2 can increase the
DNA binding activity of NF-
B through the JNK/AP-1 signaling pathway
and protect neurons from DA-induced apoptosis.
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Fig. 10.
A JNK antisense oligonucleotide
down-regulates the binding activity of AP-1 and
NF- B. Panel A, Western blot
analysis of phosphorylated JNK after treatment with JNK1/2 antisense
(AS). Cerebellar granule neurons were treated with 20 µM
Grx2 in the presence or absence of JNK1/2 antisense and sense (S)
oligonucleotides (1 µM) for 2 h. Total cellular
proteins were extracted and immunoreacted with anti-phospho-JNK
antibody, as described under "Materials and Methods." Panels
C and E, EMSA showing AP-1 and NF-
B binding activity
in nuclear extracts from cerebellar granule neurons incubated with 20 µM Grx2 for 1 h (AP-1) and 2 h (NF-
B), in
the presence or absence of JNK1/2 antisense and sense oligonucleotides
(1 µM) for 2 h. Identical cultures were incubated in
serum-free medium and served as control. Panels B,
D, and F, quantitative analysis of JNK levels,
AP-1 and NF-
B binding (respectively) are represented as a percent of
untreated cells (n = 3). ***, p < 0.001; **, p < 0.025; *, p < 0.05;
···, p < 0.001; ··,
p < 0.025 versus Grx2 activation.
Error bars represent ± S.D. Statistical analyses were
performed with a two-tailed Student's t test. C,
control.
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Fig. 11.
Grx2 neuroprotective activity is attenuated
by a JNK1/2 antisense oligonucleotide. Cerebellar granule neurons
were incubated with 600 µM DA or 20 µM Grx2
plus 600 µM DA for 5 h in the presence or absence of
JNK1/2 antisense (AS) or sense (S) oligonucleotides (1 µM). Cell viability was determined by Alamar Blue assay
(n = 4). ***, p < 0.001; **,
p < 0.025. Error bars represent ± S.D. Statistical analyses were performed with a two-tailed Student's
t test. C, control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B compared with Grxs at µM
concentrations (18). Because human Grx has the same antiapoptotic
effect as E. coli Grx2 and stimulates the DNA binding
activities of NF-
B and AP-1 to the same extent, we consider that the
use of Grx2 in experiments with neurons is biologically relevant.
B. Ref-1
was essential for the function of both pathways, whereas DA was an
inhibitor leading to neuronal attrition.
B by Grx. Different stimuli that activate NF-
B
may also activate the JNK signaling cascade (e.g. tumor
necrosis factor-
, UV irradiation) (103, 104). At the same time,
several upstream activator proteins of the JNK pathway, such as MEKK1
(mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase kinase 1), Act-1 (NF-
B-activating protein), CIKS (connection
to I-
B kinase) and JNK, can activate NF-
B through I-
B kinase
(103, 105-107). Furthermore, c-Fos and c-Jun were found to interact
physically with NF-
B p65 through the Rel homology domain. This
complex exhibited enhanced DNA binding and biological function via both
the
B and AP-1 response elements (108, 109). I-
B
is essential
for maintaining basal JNK activation and for regulating the JNK-induced
TNF resistance in fibroblasts (110). The existence of proposed
cross-talk between the Ras/PI3K/Akt and JNK/AP-1 pathways is supported
further by a recent report in which thioredoxin-induced NF-
B
activation was mediated by the MEKK1/JNK pathway (111).
B signaling pathways, as AP-1
and NF-
B binding activities were attenuated by the introduction of
Ref-1 antisense. This accords with previous studies showing the
involvement of Ref-1 in the redox regulation of AP-1 and NF-
B (73-75). The positioning of Ref-1 in the signaling cascade is very likely to be downstream of PI3K but upstream of Akt (Figs. 6 and 7).
Interestingly, Akt activation is dependent on newly synthesized Ref-1.
In the presence of Ref-1 antisense, which inhibits Ref-1 transcription
and translation, Grx2 could not stimulate Akt activity. This finding
demonstrates that activation of the Ras/PI-3 kinase/Akt pathway is
dependent on alterations in gene expression. The type of interaction
between Grx2 and Ref-1 is not known. Ref-1 could interact physically
with thioredoxin in the nucleus following ionizing radiation and
thereby activate AP-1 (112). In addition to increased Ref-1 synthesis
as a means of activation, a recent study demonstrated that Ref-1
phosphorylation by casein kinase II stimulated the redox regulation of
AP-1 (12).
B activation. These pathways are regulated by newly synthesized
Ref-1, which stimulates Akt, NF-
B, and AP-1 activities. In contrast,
DA down-regulates these pathways. These results point to the critical
importance of the redox state for activation of central cellular
pathways that determine the fate of the cell.
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Fig. 12.
Proposed model for the protective action of
Grx2. Grx2 penetrates into the cerebellar granule neurons to
activate the Ras/PI3K and JNK/AP-1 signaling pathways. Grx2 elevates
the activities of Ras and PI3K, leading to increased expression of
Ref-1. Ref-1 activates Akt, which phosphorylates I- B kinase (IKK),
which in turn phosphorylates I
B
, thereby accelerating its
ubiquitination and degradation leading to NF-
B activation. In
parallel, Grx2 activates JNK and AP-1, the final effect being the
stimulation of NF-
B binding activity. Ref-1 stimulates the DNA
binding activity of both NF-
B and AP-1. DA blocks PI3K/Akt and
JNK/AP-1 pathways and attenuates NF-
B binding activity. Blue
arrows represent the activities induced by Grx2; red
arrows show the effects of DA. AS, antisense; P-, phospho-.
![]() |
FOOTNOTES |
---|
* This research was supported in part by Israel Science Foundation Grant 502/00-1, the Israeli Ministry of Health, Swedish Cancer Society Grant 961, and Swedish Medical Research Council Grant 03529.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.
** Recipient of a Sackler fellowship at Tel Aviv University.
To whom correspondence should be addressed. Tel.:
972-3-640-9782; Fax: 972-3-640-7643; E-mail:
barzilai@post.tau.ac.il.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M101400200
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ABBREVIATIONS |
---|
The abbreviations used are:
Grx, glutaredoxin(s);
DA, dopamine;
NF-B, nuclear factor-
B;
PI3K, phosphoinositide 3-kinase;
JNK, Jun N-terminal kinase;
Ref-1, redox
factor-1;
FTS, trans-farnesyl thiosalicylic acid;
DTT, dithiothreitol;
PBS, phosphate-buffered saline;
DAPI, 4,6-diamidino-2-phenylindole;
EMSA, electrophoretic mobility shift
assay;
GST-RBD, glutathione S-transferase fused to the Ras
binding domain of Raf-1.
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