From the Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, and the § Department of Human
Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv
University, Tel Aviv 69978 Israel
Received for publication, October 31, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Maintenance of genome stability is essential for
keeping cellular homeostasis. The DNA damage response is a
central component in maintaining genome integrity. Among of the most
cytotoxic DNA lesions are double strand breaks (DSBs) caused by
ionizing radiation or radiomimetic chemicals. ATM is missing or
inactivated in patients with ataxia-telangiectasia.
Ataxia-telangiectasia patients display a pleiotropic phenotype and
suffer primarily from progressive ataxia caused by degeneration of
cerebellar Purkinje and granule neurons. Additional features are
immunodeficiency, genomic instability, radiation sensitivity, and
cancer predisposition. Disruption of the mouse Atm locus
creates a murine model of ataxia-telangiectasia that exhibits most of
the clinical features of the human disease but very mild neuronal
abnormality. The ATM protein is a multifunctional protein kinase, which
serves as a master regulator of cellular responses to DSBs. There is
growing evidence that ATM may be involved in addition to the DSB
response in other processes that maintain processes in cellular
homeostasis. For example, mounting evidence points to increased
oxidative stress in the absence of ATM. Here we report that the AP-1
pathway is constantly active in the brains of Atm-deficient mice not
treated with DNA damaging agents. A canonical activation (increased
phosphorylation of mitogen-activated protein kinase kinase-4,
c-Jun N-terminal kinase, and c-Jun) of the AP-1 pathway was found in
Atm-deficient cerebra, whereas induction of the AP-1 pathway in
Atm-deficient cerebella is likely to mediate elevated expression of
c-Fos and c-Jun. Although Atm+/+ mice are capable of
responding to ionizing radiation by activating stress responses such as
the AP-1 pathway, Atm-deficient mice display higher basal AP-1 activity
but gradually lose their ability to activate AP-1 DNA-binding activity
in response to ionizing radiation. Our results further
demonstrate that inactivation of the ATM gene
results in a state of constant stress.
Ataxia-telangiectasia
(A-T)1 is an autosomal
recessive disorder characterized by progressive cerebellar ataxia,
immunodeficiency, premature aging, gonadal dysgenesis, extreme
radiosensitivity, and a high incidence of lymphoreticular malignancies
(for review, see Ref. 1). Functional inactivation of the ATM
gene product accounts for this complex phenotype (2). The ATM protein,
the product of the ATM gene, is a member of a family of
large protein kinases found in various organisms, which share
phosphatidylinositol 3 kinase-like domain. ATM is a serine/threonine
protein kinase that mediates the activation of multiple signaling
transduction pathways following the induction of DNA double strand
breaks (DSBs) (reviewed in Refs. 3 and 4).
Atm-null mice that were created by disrupting the Atm locus
(5-8) recapitulate most of the features of the human A-T phenotype and
display growth retardation, male and female infertility, extreme predisposition to thymic lymphomas, and acute sensitivity to ionizing radiation (IR). Of note, the neurological symptoms of the disease are
very mild in the animal model and can be observed only after challenge
(5, 8, 9).
Cells derived from A-T patients and Atm-deficient mice exhibit genomic
instability, extreme sensitivity to DNA breaking agents, and a broad
defect in the activation of the cell cycle checkpoints and other
pathways induced by DSBs (3, 10). However, A-T cells are not sensitive
to UV light or to replication inhibitors. To activate the DNA damage
response, ATM phosphorylates a number of downstream targets, including
p53, Mdm2, Brca1, Chk2, and Nbs1 (reviewed in Refs. 3 and 4). ATM is
activated by DSBs induced by internal or external DNA damaging agents,
such as IR, radiomimetic chemicals, and reactive oxygen species and is
probably activated also by DSBs that occur normally during meotic and
V(D)J recombination. ATM can bind to DNA in vitro (11, 12),
and a fraction of ATM binds tightly to the chromatin following the
induction of DSBs, in accordance with the suggested role of ATM in the
early steps of the DNA damage response.
Evidence is accumulating that ATM is also involved in maintaining
genomic stability under normal conditions of apparently no stress.
Because it appears to mediate IR-induced death in the murine-developing
nervous system (13), it was hypothesized that it functions during
nervous system development to eliminate neuronal cells afflicted with
genomic damage (13). ATM was also shown to be required for the
execution of apoptosis in all areas of the developing nervous
system in a model of a DNA ligase IV-deficient mouse (14).
One of the major stress responses is mediated by the AP-1 transcription
factor. AP-1 is a superfamily of proteins consisting of several
subfamilies, including Jun, Fos, and ATF-2, all of which possess a
leucine zipper domain that assists their dimerization (15). The AP-1
proteins form homo- and heterodimers before binding to their DNA target
sites. The activation of AP-1 is regulated by c-Jun N-terminal kinase
(JNK), ERK, and p38 MAPKs (mitogen-activated protein kinases) (16, 17).
JNKs are a family of serine-threonine kinases related to the MAPKs with
at least 10 isoforms encoded by three genes (JNK1,
JNK2, and JNK3), with additional diversification resulting from alternative splicing (18-28). The JNK1 and JNK2 subfamilies each comprise four isoforms (46 or 55 kDa), whereas the
JNK3 contains two different isoforms (45-48 or 54-57 kDa). Although
JNK1 and JNK2 are expressed in most cell types (23), expression of JNK3
is limited to neuronal cells (18). All JNKs possess a conserved TPY
tripeptide motif in their kinase domain (Thr-183 and Tyr-185)
(22), and their activation is mediated by phosphorylaion of the
threonine and tyrosine residues within this motif by the upstream JNK
kinases MKK4 (29-31) and MKK7 (32). Among the known subsrates of the
JNKs are the c-Jun protein, a component of the AP-1 transcription
factor (33), and the transcription factors ATF-2 (34), Elk1 (35), and
p53 (36). Although all JNKs are capable of phosphorylating c-Jun on
serine residues 63 and 73, isoforms exhibit different substrate-binding
specificities (37, 38).
In vitro studies have revealed a defect in IR-induced
activation of the JNK-signaling pathway in lymphoblastoid cells from individuals with A-T. Although IR induced c-Jun phosphorylation in
normal cells, no such phenomenon was observed in A-T fibroblasts. The
46-kDa isoform of JNKs was dually phosphorylated in the nuclei of both
normal and A-T fibroblast following exposure to IR, whereas c-Jun activity was detected in normal cells but not in A-T fibroblasts. Furthermore, an exogenous-purified active JNK protein was able to
phosphorylate endogenous c-Jun in nuclear extracts only of normal but
not A-T cells and only after the normal cells were irradiated, (39).
Here we report that the AP-1 pathway is constantly active in untreated
Atm-deficient brain. Atm+/+ mice are capable of responding
to IR by activating the AP-1 pathway, whereas Atm-deficient mice
display higher basal AP-1 activity but gradually lose their ability to
activate AP-1 DNA-binding activity in response to IR.
Mice--
Atm+/ Total Protein Extracts--
Tissues (cerebellum and cerebrum)
were washed with ice-cold phosphate-buffered saline and homogenized in
ice-cold homogenation buffer A (150 mM NaCl, 50 mM Tris-HCl, pH 7.6, 10% glycerol, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor
mixture (Roche Molecular Biochemicals), 0.2 mM
NaVO3, 0.1 mg/ml leupeptin (400 µl for cerebellum and 1.5 ml for cerebrum)). The lysates were sonicated and then centrifuged for
5 min at 2,000 × g. The pellets were removed and the
supernatants were collected and frozen.
Nuclear Extracts--
Tissues (cerebellum, cerebrum, and liver)
were washed with ice-cold phosphate-buffer saline and maintained in
0.32 M sucrose. The organs were homogenized in ice-cold
hypotonic buffer B (0.5 M sucrose, 10 mM HEPES
pH 7.9, 1.5 mM MgCl2, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1 mM
PMSF and protease inhibitor mixture) and 0.2 mM
Na3VO4. The lysates were allowed to
incubate for 15 min on ice and then centrifuged for 10 min at
7,500 × g. The supernatants were removed (cytoplasmic
extracts), and the nuclear pellets were rinsed once with ice-cold
buffer B and resuspended in buffer C (0.42 M NaCl, 20 mM Tris-HCl, pH 7.9, 25% glycerol, 0.2 mM
EDTA, 1 mM dithiothreitol, and protease inhibitor mixture
(Roche Molecular Biochemicals), 0.2 mM NaVO3
(100 µl for cerebellum, 400 µl for cerebrum, and 0.5 ml for
liver)). The nuclear extracts were incubated for 45 min on ice and
centrifuged for 20 min at 15,500 × g. The supernatants
were collected and used for electrophoretic mobility shift assay (EMSA)
and Western blot analyses.
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
AP-1 oligonucleotides (5'-CGCTTGATGAGTCAGCCGGAA-3', respectively)
(Promega, Madison, WI) was incubated for 30 min with 6 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, Sunnyvale, CA). Quantitative data were obtained
using phosphoimaging analysis (Molecular Dynamics).
Western Blotting--
Western blotting was performed as
described by Harlow and Lane (61), using 12.5% polyacrylamide
gels (PAGE). Each lane was loaded with an identical amount of protein
extract (50 or 100 µg), which, following electrophoresis, were
transferred to an Immobilon polyvinyldene disulfide membrane for
1.5 h. Blots were stained with Ponceau to verify equal loading and
transfer of proteins. Membranes were then probed with anti-phospho-JNK
(1:1000) antibodies (Cell Signaling Technology, Beverly, MA), mouse
anti-c-Jun (1:1000) antibodies (BD Biosciences), mouse
anti-c-Fos (1:400) antibodies (Santa Cruz, CA), mouse
anti-phospho-c-Jun (Ser-63) (1:1000) antibodies (Santa Cruz
Biotechnology), and rabbit anti-phospho-MKK4 (1:1000) antibodies (Cell
Signaling Technology, Beverly, MA). For the detection of phospho-JNK
and phospho-c-Jun, EnVisionTM+/horseradish
peroxidase (rabbit, 1:100) (DAKO, Glostrup, Denmark) was used to
enhance sensitivity. The rest of the antibodies were detected using
horseradish peroxidase-conjugated antibodies (1:25000 for mouse and
1:15000 for rabbit) purchased from Jackson Laboratories. Intensity of the signal was determined by the ECL-Plus detection system
(Amersham Biosciences). Protein concentration was determined according
to the method of Bradford (62) using BSA as standard.
Statistical Analysis--
Results were analyzed using two-tailed
Student's t test. Data were expressed as mean ±S.E., and
p values Atm Deficiency Leads to Gradual Induction of AP-1 DNA-binding
Activity--
The AP-1-signaling pathway is one of the most prominent
pathways known to respond to various stress conditions. Alterations in
the activity of this pathway in a particular tissue imply that it is
under stress. To test whether tissues of Atm-deficient mice are under
constant stress, we analyzed the basal level of AP-1 DNA-binding
activity in the brains of Atm
The nervous system, especially the cerebellum, is highly
affected in A-T; other organs such as the liver are not known to be
involved. This coincides with our earlier findings that the cerebellum
but not the liver is under constant oxidative stress (40). To correlate
the degree of stress to AP-1 DNA-binding activity, we measured that
activity in livers isolated from 4-month-old Atm Atm Loss Leads to Constant Induction of JNK Activity--
The
earlier step in the signaling pathway that leads to AP-1 induction is
JNK-mediated phosphoryaltion of the N terminus of c-Jun. This
phosphorylation activates its dimerization (either with Jun or Fos
isoforms) and binding to its target consensus sequence. To test whether
Atm loss leads to increased JNK activity, we measured the levels of
phospho-JNK, the activated form of this enzyme (Fig.
2). There was a gradual increase in
phospho-JNK levels in Atm-deficient brains, from no change compared
with normal tissue at the age of 1 month to close to 3-fold induction
at 4 months (p < 3.05 × 10 Elevated Phospho-MKK4 Levels in Atm-deficient
Cerebra--
MKK4 is among the kinases capable of activating
JNK. To explore whether Atm loss leads to constant activation of MKK4,
we examined the levels of activated MKK4 in 4-month-old cerebra and cerebella. Interestingly, we found elevation (67% increase,
p < 0.03) only in Atm-deficient cerebra and not in the
cerebella (Fig. 3).
Atm Atm Atm-deficient Cerebra Display High Basal Levels and
Irradiation-induced Elevation of
Ser(P)-63-c-Jun--
Atm-deficient mice were capable of
activating JNK in response to IR but failed to induce AP-1 DNA-binding
activity. To test whether Atm loss disrupts the ability of JNK to
phosphorylate its target proteins, we analyzed the levels of
phospho-c-Jun in the cerebra and cerebella of untreated and irradiated
animals. Using antibody directed against c-Jun phosphorylated on
Ser-63, we observed a 50% increase in phospho-c-Jun levels
(p < 0.017) in cerebra of 4-month-old untreated
Atm-deficient mice. In contrast, no change in Ser(P)-63-c-Jun levels
was observed in Atm-deficient cerebella (Fig.
6, upper panel). Exposing the
animals to 20 Gy x-ray resulted in a gradual increase (2-3-fold) in
Ser(P)-63-c-Jun levels only in the cerebra of 1-month- and 4-month-old
Atm+/+ mice, but not in the same tissues of
Atm Increased c-Fos and c-Jun Expression in Untreated Atm-deficient
Cerebella--
Elevated levels of either c-Jun or c-Fos proteins can
lead to constant AP-1 activation. To test whether Atm deficiency leads to alterations in c-Jun and c-Fos levels, we analyzed the basal levels
of these proteins in untreated Atm-deficient cerebella and cerebra.
Using antibody directed against c-Fos, we observed a more than 3-fold
increase in c-Fos levels (p < 0.019) in cerebella of
4-month-old untreated Atm-deficient mice compared with
Atm+/+ animals. Smaller induction in c-Fos levels
(1.8-fold, p < 0.032) was observed in the cerebra.
Elevated levels of c-Jun were observed only in 4-month-old untreated
Atm-deficient cerebella (2.5-fold, p < 0.0242). No
change in c-Jun levels was observed in Atm-deficient cerebra (Fig.
7).
Alterations in the activity of the AP-1-signaling pathway in
a given tissue imply that the tissue may be under stress. The data
reported here, together with other reports (40-43), further substantiate the facts that organs most affected in A-T patients gradually develop stress conditions. Several of our findings support this notion: (a) Atm-deficient brains, but not Atm-deficient livers, display higher basal AP-1 DNA-binding activity. (b) Untreated Atm-deficient cerebra and cerebella display elevated levels of phospho-JNK. (c) Atm-deficient mice gradually lose their ability to
activate AP-1 DNA-binding activity in response to IR, suggesting that
the system is either saturated and can no longer respond to stress
stimuli, or accumulated damage disrupts the signaling pathways
mediating radiation-induced AP-1 activation. (d) There are increased
levels of phospho-c-Jun in untreated Atm-deficient cerebra. (e) There
are elevated levels of c-Fos in untreated Atm-deficient cerebella. Our concept that Atm loss leads to constant stress in
organs most affected in A-T is further supported by our recent findings
using microarray DNA chips. Initial analysis revealed that unirradiated
Atm-deficient cerebella displayed constitutive activation of repression
of numerous genes that the corresponding wild type cerebella showed
only after irradiation.2
AP-1 DNA-binding activity can be induced in two ways: by
increased transcription of c-Jun or Fos, or by phosphorylation of the Jun proteins, mainly by different JNK isoforms (for review see Ref.
44). Canonical AP-1 activation was observed in the cerebra of untreated
Atm-deficient mice, but there was no c-Jun phoshorylation either on
Ser-63 or Ser-73 in the cerebella (see Fig. 6). In untreated
Atm-deficient cerebra, elevated levels of phospho-MKK4, JNK, and
Ser(P)-63-c-Jun were observed, whereas higher levels of c-Fos and c-Jun
were likely to mediate Atm-induced AP-1 activation in the cerebella.
Interestingly, DNA microarray analysis revealed a significant elevation
of another member of the Fos family, FosB, in untreated Atm-deficient
cerebella, suggesting that Fos proteins play an important role in
cellular processes induced by Atm deficiency. Increased and prolonged
expression (mRNA and protein) of c-Jun and c-Fos with no apparent
elevation in c-Jun (Ser-63) phosphorylation was observed in the
hippocampal CA1 pyramidal cell layer in regions undergoing delayed
neuronal death (45). Similar to what we observed in Atm-deficient
cerebella, these results demonstrate that increased AP-1 DNA-binding
activity can stem solely from an increase in c-Fos and c-Jun. The
biological role of JNK activation in Atm-deficient cerebella remains
unclear because none of its downstream substrates were found to be
elevated. It is possible that JNK activation is merely a consequence of Atm loss with no actual biological role. Taken together, our results indicate that the cerebella and the cerebra are different entities with
regard to AP-1 activation.
The AP-1 pathway can be activated in response to diverse stimuli and a
wide range of stresses. However, its role in stress remains largely
unknown because both pro-apoptotic and pro-survival effects of AP-1
have been observed (for reviews see Refs. 46 and 47). DNA damage,
especially DSBs, poses a critical threat to the cell. The finding of an
accumulation of DNA breaks in Atm-deficient cerebella with no signs of
cell death (48) argues in favor of a protective role for AP-1. Our
data, on the other hand, does not rule out a pro-apoptotic role for
AP-1. A pro-survival role for AP-1 activity is also consistent with
several reports showing that AP-1 activity is involved in defense
against UV radiation and DNA-alkylating agents (49). AP-1 was shown to
be involved in DNA repair by its ability to regulate the expression of
O6-methylguanine-DNA methyltransferase; the
promoter of this gene was found to contain two putative sites of AP-1
(50). This enzyme is up-regulated in response to a variety of genotoxic
insults (for review see Ref. 51). Stable expression of a
non-phosphorylated dominant negative protein c-Jun (S63A, S73A)
markedly inhibited AP-1 activity and greatly increased the cytotoxic
effects of a variety of DNA-damaging agents (52). In contrast to the
previous reports, DNA fragmentation and activation of c-Jun in
cerebellar neuronal cells can lead to their demise. Gillardon et
al. (53) found increased DNA fragmentation and c-Jun up-regulation
in Purkinje cells of weaver and pcd mutant mice. Weaver and
Purkinje cell degeneration (pcd) are autosomal mutations
that lead to complete loss of granule and Purkinje cells, respectively.
Co-localization of DNA fragmentation and c-Jun in dying granule cells
(weaver) and Purkinje cells (pcd), and inhibition of the
death process by function-blocking c-Jun antibodies, led Gillardon
et al. (53) to suggest a pro-apoptotic role for c-Jun. One
way to explain the dual role (death or survival) of AP-1 is by their
ability to form different dimer combinations. A 35-kDa Fos-related
antigen/JunD dimer was detected in neurons that survived injury,
whereas a Jun·JunD complex was detected in neurons prior to
undergoing apoptosis (54). Because the present study was carried out
for the whole brain, it is not clear which of the AP-1 complexes
actually functions in specific cell populations.
A prolonged induction of phospho-JNK and AP-1 was observed in
Atm-deficient cerebella and cerebra (Figs. 1 and 3). Prolonged expression of some AP-1 components, such as Fos-related antigen 2, was
associated with neuronal regeneration and repair (55). Following this
line of thinking, it is possible that prolonged AP-1 induction in
Atm-deficient brains is important for neuronal survival. Our results
are consistent with several studies (56, 63) showing prolonged AP-1
activation in Atm-deficient cells. Whereas wild type fibroblast
showed transient induction of c-Jun in response to IR, ATM-deficient
fibroblasts responded to IR with prolonged c-Jun expression. Lee
et al. (56) showed delayed and prolonged JNK activation in
response to IR and oxidative stress.
Increased levels of phospho-JNK can stem from two opposing
processes: elevated activity of upstream kinases such as MKK4/7, or
reduced activity of phosphatases such as MAPK phosphatase-5 (MKP-5).
Bar-Shira et al. (57) showed that treatment with the radiomimetic agent neocarzinostatin resulted in elevated levels of
MKP-5 in wild type cells, which was attenuated in ATM-deficient human
cells. Moreover, the basal level of this phosphatase was lower in
ATM-deficient cells. These findings support our hypothesis that
Atm-deficiency leads to accumulation of DNA breaks especially in the
cerebellum (48), where Atm loss retards the induction of MKP-5 and
thereby increases the level of phospho-JNK in untreated Atm-deficient
cerebella. In the presence of Atm, DNA damage will lead to a timely
controlled cycle of phosphorylation and dephosphorylation of MAPKs; the
activation of this cycle is defective in Atm-deficient cells,
accounting for the defective ability of these cells to generate correct
stress responses. Chen et al. (58) showed that increased
oxidative stress induced JNK activity, not by activating its upstream
kinases stress-activated protein kinase/ERK kinase (SEK1)/MKK4
or MKK7, but rather by inactivating its phosphatase M3/6. These
results are also consistent with our notion that prolonged AP-1
activation stems from DNA damage-induced oxidative stress (40, 43, 48).
DNA damage can activate the AP-1 pathway regardless of oxidative stress.
Using whole cerebral and cerebellar extracts precluded our determining
the exact cellular population in which AP-1 is up-regulated in
untreated Atm-deficient mice. Besides neurons, a variety of distinct
cell populations can activate AP-1 pathways. Masood et al.
(59) showed that AP-1 is an important transcription regulator of the
astrocyte specific gene, glial fibrillary acidic protein (GFAP), which
is up-regulated in proliferating and activated astrocytes. Our
observation of elevated levels of GFAP in the cerebella of Atm-deficient mice3 further
supports the notion that AP-1 is up-regulated in astrocytes. Proliferation and activation of astrocytes and microglia cells may
contribute to an inflammatory reaction as a result of tissue damage.
Our failure to detect increased microglia proliferation or activation
argue against cerebral or cerebellar inflammation. It is possible that
astrocytes were activated in response to increased oxidative stress,
which has been shown to develop in untreated Atm-deficient mice
especially in the cerebella. Astrocytes were indeed found capable of
altering their phenotype with up-regulation of large number of
molecules, including those controlling the protective system (for
review see Ref. 60).
In summary, we have demonstrated that ATM loss leads to constant
activation of the AP-1-signaling pathway in untreated Atm-deficient brains in an age-dependent manner. Moreover, Atm-deficient
mice gradually lose their ability to activate the AP-1-signaling
pathway in response to IR. Based on these findings, we suggest that
accumulation of unrepaired DNA breaks exerts constant stress on the DNA
damage response mechanisms, whose overactivation leads to constant
activation of critical signaling pathways, ultimately disturbing
cellular homeostasis. Better understanding of the ongoing stress
responses in Atm-deficient tissues may point to novel therapeutic
approaches to help alleviate some of the symptoms associated with A-T,
in particular the progressive neurodegeneration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mice (5) were a generous gift from
Dr. Wynshaw-Boris. Offspring of these mice were genotyped by PCR-based
assays using mouse-tail DNA, prepared with the
GeneReleaserTM reagent (Bio-Ventures Co., Murfreesboro, TN).
0.05 were considered significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
- and
Atm+/+-untreated mice. In 1-month-old animals, the
differences in AP-1 activity between Atm
/
and
Atm+/+ cerebella and cerebra were significant but rather
small (45% induction in Atm
/
animals,
p < 0.025 for the cerebellum and p < 0.0023 for the cerebrum). In 4-month-old animals, there was a 3-fold
constant induction of AP-1 activity in Atm-deficient tissues
(p < 0.0079 for the cerebellum and p < 0.0264 for the cerebrum) (Fig.
1A).
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Fig. 1.
AP-1 DNA-binding activity in Atm-deficient
brains and livers. EMSA showing AP-1 DNA-binding activity in
nuclear extracts from 1-month-old (upper panel) and
4-month-old-old (lower panel) cerebellum and cerebrum
(A) and 4-month-old livers isolated from
Atm /
- and Atm+/+-untreated mice
(B). Note the gradual increase in AP-1 DNA-binding activity
in Atm-deficient brain. Quantification of band intensities was
performed using TINA software. ***, p < 0.01, **,
p < 0.025, *, p < 0.05. Error
bars represent ±S.E. Statistical analyses were performed with
two-tailed Student t test (n = 3 for
1-month-old, n = 7 for 4-month-old mice brains, and
n = 4 for the livers).
/
- and
Atm+/+-untreated animals. Atm-deficient mice and their
control littermates displayed similar levels of AP-1 DNA-binding
activity (Fig. 1B). Taken together, the results presented in
Fig. 1 show that organs most affected in A-T display higher levels of
AP-1 DNA-binding activity, whereas non-affected organs behave like controls.
5 for
the cerebellum and p < 0.0037 for the cerebrum). The
level of JNK induction correlates well with the induction of AP-1
DNA-binding activity (compare Figs. 1 and 2).
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Fig. 2.
Up-regulation of phospho-JNK levels in
Atm-deficient mice. Nuclear extracts were isolated from the
cerebrum and cerebellum of 1-month-old (upper panel) and
4-month-old (lower panel) Atm /
and
Atm+/+, separated on 12.5% polyacrylamide gel, and blotted
onto polyvinyldene disulfide membrane. The membrane was stained with
Ponceau, after which the blot was reacted with anti-phospho-JNK
polyclonal antibody (1:1000). The blots were developed using the ECL
system, and a representative Western blot is shown. To verify that
equal amounts of protein were transferred, the blots were stripped and
reacted with mouse monoclonal anti-tubulin antibody (1:25,000).
Quantitative analysis of bands was performed using TINA software and
normalized to the corresponding tubulin. Quantitative analysis of bands
is represented by arbitrary units Atm+/+ mice
(n = 10).
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Fig. 3.
Up-regulation of phospho-MKK4 levels in
Atm-deficient cerebra. Nuclear extracts were isolated from the
cerebrum and cerebellum of 4-month-old Atm /
and
Atm+/+ and treated as described in the legend to Fig. 2.
The blot was reacted with anti-phospho-MEK4 polyclonal antibody
(1:1000). The blots were developed using the ECL+ system. Verification
of equal loading and quantitative analysis were performed as described
in the legend to Fig. 3 (n = 8).
/
Mice Gradually Lose their Ability to Activate
AP-1 DNA-binding Activity in Response to IR--
Atm
/
mice and their control littermates were exposed to 20 Gy of x-rays for
different time periods, after which the levels of AP-1 DNA-binding
activity were assayed. At 4 months of age, Atm+/+ brains
responded to the irradiation treatment with 2.5-fold induction in AP-1
DNA-binding activity (Fig 4). A different
pattern of AP-1 induction was observed between Atm+/+
cerebellum and cerebrum. The Atm+/+ cerebellum displayed a
clear peak of AP-1 induction around 60 min post-irradiation, whereas
the cerebrum showed a rapid and steady induction in AP-1 levels. In
contrast, 4-month-old Atm
/
mice failed to activate AP-1
DNA-binding activity in response to IR, and the levels of
AP-1 remained unchanged (Fig. 5,
right panel). Interestingly, young Atm
/
mice
retained their ability to induce AP-1 DNA-binding activity in response
to IR (Fig. 4, left panel). Similar to Atm+/+,
2-fold induction in AP-1 DNA-binding activity was measured in these
Atm
/
cerebella and cerebra. These results suggest that
damage accumulation is gradually accompanied by loss of the ability of
Atm-deficient mice to activate stress responses as a result of noxious
stimuli such as IR.
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Fig. 4.
Alterations in AP-1 DNA-binding activity in
Atm /
and Atm+/+ brains following exposure
to 20 Gy x-ray. Upper panel, EMSA showing AP-1
DNA-binding activity in nuclear extracts from 1-month-old (left
panel) and 4-month-old (right panel) cerebellum and
cerebrum isolated from Atm
/
- and
Atm+/+-irradiated and untreated mice. The mice
(n = 2 for each time point) were irradiated (20 Gy) and
samples taken at the indicated time points. Lower panel,
quantification of band intensities was performed using TINA software.
Quantitative analysis of AP-1 induction is represented as a fold of
induction of AP-1 in Atm
/
tissues compared with
Atm+/+-untreated mice. Error bars represent ± S.D.
, Atm+/+ cerebellum;
, Atm
/
cerebellum;
, Atm+/+ cerebrum; and
,
Atm
/
cerebrum.
View larger version (52K):
[in a new window]
Fig. 5.
Alterations in phospho-JNK (P-JNK)
levels in Atm /
and Atm+/+ brains
following exposure to 20 Gy x-ray. Nuclear extracts were isolated
from the cerebrum and cerebellum of 1-month-old (upper
panel) and 4-month-old (lower panel) irradiated and
untreated Atm
/
and Atm+/+ mice. The
extracted protein was separated on 12.5% polyacrylamide gel and
blotted onto polyvinyldene disulfide membrane and treated as indicated
in the legend to Fig. 2. The animals (n = 2 per each
time point) were irradiated (20 Gy) and samples taken at the indicated
time points. Quantitative analysis of bands was performed using TINA
software and normalized to the corresponding tubulin.
,
Atm+/+ cerebellum;
, Atm
/
cerebellum;
, Atm+/+ cerebrum; and
, Atm
/
cerebrum. Quantitative analysis of bands is represented by arbitrary
units Atm+/+-untreated mice (n = 6-8).
/
Mice Are Capable of Activating JNK in Response
to IR--
Next we assayed the ability of Atm
/
and
Atm+/+ mice to activate JNK in response to 20 Gy x-ray.
Similar results were obtained in younger (1-month-old) and older
(4-month-old) Atm
/
and Atm+/+ mice. In both
cases, the peak of phospho-JNK induction was around 60 min
post-irradiation (Fig. 5). Four-month-old Atm-deficient mice are
incapable of activating AP-1 DNA-binding activity, but notably they
were capable of elevating phospho-JNK levels in response to IR to a
level similar to their control littermates. These results suggest that
accumulating damage may not affect JNK activity in response to IR but
rather events downstream to it.
/
mice.
View larger version (27K):
[in a new window]
Fig. 6.
Alterations in Ser(P)-63-c-Jun levels in
Atm /
- and Atm+/+-untreated brains and
following exposure to 20 Gy x-ray. Nuclear extracts were isolated
from the cerebrum and cerebellum of 1-month-old (upper
panel) and 4-month-old (lower panel) irradiated and
untreated Atm
/
and Atm+/+ mice. The
extracted proteins were separated on 12.5% polyacrylamide gel and
blotted onto polyvinyldene disulfide membrane and reacted with
anti-phospho-c-Jun (Ser-63) antibodies. Mice (n = 2 per
each time point) were irradiated with a dose of 20 Gy. Quantitative
analysis of bands was performed using TINA software and normalized to
the corresponding tubulin. The quantitative analysis of bands is
represented as fold induction of Atm+/+-untreated mice.
Error bars represent ± S.D.
, Atm+/+
cerebellum;
, Atm
/
cerebellum;
,
Atm+/+ cerebrum; and
, Atm
/
cerebrum
(n = 8).
View larger version (14K):
[in a new window]
Fig. 7.
Alterations in c-Fos and c-Jun levels in
Atm /
- and Atm+/+-untreated brains.
Nuclear extracts were isolated from the cerebrum and cerebellum of
4-month-old untreated Atm
/
and Atm+/+ mice.
The extracted protein was separated on 12.5% polyacrylamide gel,
blotted onto polyvinyldene disulfide membrane, and reacted with either
c-Fos (left panel) or c-Jun (right panel)
antibodies. Quantitative analysis of bands was performed using TINA
software and normalized to the corresponding tubulin. The quantitative
analysis of bands is represented as fold induction of
Atm+/+-untreated mice. **, p < 0.025, *, p < 0.05. Error bars represent
±S.E. Statistical analyses were performed with two-tailed Student
t test (n = 6-8).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Yechiel Leser and Dani Schifter for valuable help.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Ataxia-Telangiectasia Children's Project (to A. B. and Y. S.), a grant from the Ataxia-Telangiectasia Medical Research Foundation (to Y. S.), and the Israel Science Foundation Grant 502/00-1 (to A. B.).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.
¶ 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, December 19, 2002, DOI 10.1074/jbc.M211168200
2 S. Rashi-Elkeles, A. Regev, R. Elkon, R. Sharan, N. Zak, L. Brodsky, A. Kamsler, A. Leontovitch, N. Weizman, D. Leshkovitz, O. Mor, A. Barzilai, E. Feinstein, R. Shamir, Y. Shiloh, and A. Bar-Shira, unpublished results.
3 N. Weizman, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: A-T, ataxia-telangiectasia; DSB, double strand break; IR, ionizing radiation; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; EMSA, electrophoretic mobility shift assay.
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