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INTRODUCTION |
In recent years, members of the c-Jun N-terminal kinase
(JNK)1 family of
mitogen-activated protein kinases have emerged as important players in
neurodegenerative disorders, such as Alzheimer's (1) and Parkinson's
disease (2), as well as in cellular stress responses (Ref. 3; reviewed
in Ref. 4).
The JNK family consists of the ubiquitously expressed JNK1 and JNK2 and
of JNK3, which is primarily expressed in the heart, brain, and testis
(5, 6). Differential splicing and exon usage yield a total of 10 different isoforms. After activation, JNKs phosphorylate various
substrates in the nucleus, e.g. c-Jun or activating
transcription factor-2 (ATF-2), and in the cytoplasm, e.g.
Bcl-2, neurofilaments or tau (6, 7).
JNKs were first described as stress kinases, and it is their
involvement in the response to cellular stress that has been studied
extensively (6, 8, 9). Especially JNK3 has mostly been associated with
apoptosis in pathological contexts and, because of its defined
expression, predominantly with neuronal apoptosis. For example,
disruption of the mouse JNK3 gene caused resistance to the excitotoxic
glutamate receptor agonist kainic acid, with reduction in seizure
activity and apoptosis of hippocampal neurons (10). JNK3 is likely to
contribute to ischemic death in perinatal and adult rats (11, 12). In
cortical and cerebellar neurons, JNK3 holds a central role in apoptosis
caused by UV irradiation,
-amyloid, growth factor withdrawal, and
sodium arsenite (13-16) or at least a partial role in cell death
following DNA damage (17). Finally, a crucial function has been
attributed to JNK3 in the induction of p75-mediated apoptosis in
oligodendrocytes (18).
Thus, the majority of reports indicate a pro-apoptotic role for JNKs.
Their function, however, is clearly not restricted to apoptosis.
Already at the beginning of JNK research, a dichotomous role for
activated JNKs and their phosphorylated substrate c-Jun was suggested
in neuronal injury connecting cell death with the competence for
regeneration (19-21). Numerous reports attribute JNKs functions in
neurite outgrowth in vitro (22-26), in the neuronal response to growth factors (27, 28), and in regeneration in vivo (21, 29). The wide range of JNK actions is further emphasized by the observation that within a single cell, different subpools of
JNKs have distinct functions (3). In summary, JNKs have an extensive
potential to perform different actions. However, the contribution of
individual isoforms is still obscure.
During the last decades, the rat pheochromocytoma cell line PC12 has
been established as a model system for neuronal apoptosis as well as
for neuronal differentiation and neurite outgrowth in response to NGF.
Therefore, PC12 cells are the system of choice for investigating the
potential of JNKs in the context of neuronal apoptosis and
differentiation. Importantly, PC12 cells only express JNK1 and JNK2
(30, 31), and these qualities render PC12 cells into a well defined
cell culture system for the analysis of transfected JNK3.
In the present study, we have investigated the capacity of a single
JNK3 isoform for the propagation of cell death and differentiation. We
demonstrate that the expression of JNK3 promotes cell death after UV
irradiation and application of taxol but not after
H2O2. On the other hand, however, transfected
JNK3 significantly increases NGF-induced neurite outgrowth. Concomitant
with the shift of function is the switch of JNK3 substrates, changing
from ATF-2 in the apoptotical context to c-Jun during neuronal
differentiation. Our results not only shed new light on the role of
ATF-2 in the context of cell death but also define a model case for the
dichotomous role of JNKs in coupling neuronal cell death and
regeneration (32).
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EXPERIMENTAL PROCEDURES |
Materials--
The following materials were used: anti-active
JNK (Promega), anti-c-Jun (Santa Cruz), anti-mouse IgG (Amersham
Biosciences), anti-neurofilament (Sigma), anti-phospho-ATF-2 and
anti-phospho-c-Jun (Cell Signaling Technologies), pEGFP-C3 vector
(Clontech), ECL detection system (Amersham
Biosciences), fetal calf serum (Bio Whittaker),
H2O2 (Sigma), Geneticin (G418) (Stratagene),
horseradish peroxidase-linked donkey anti-rabbit antibody (Cell
Signaling Technologies), horse serum (Invitrogen), Hyperfilm (Amersham
Biosciences), Immobilon-P (Millipore), paclitaxel/taxol (Calbiochem),
penicillin/streptomycin solutions (Invitrogen), phosphatase inhibitor
mixture II (Sigma), Ponceau S (Sigma), recombinant mouse NGF (2.5 S)
(alomone labs), protease inhibitor (complete) (Roche Molecular
Biochemicals), RPMI 1640 medium (Invitrogen), TransFast transfection
(Promega), and trypan blue stain (Sigma).
Cell Culture--
Rat pheochromocytoma PC12 cells were cultured
on collagen-coated plates in RPMI 1640 medium supplemented with 5%
heat-inactivated calf serum, 10% heat-inactivated horse serum, and 1%
penicillin/streptomycin at 37 °C and 5% CO2.
Transfected cell clones were kept in the presence of 500 µg/ml G418
to maintain selection.
Plasmids and Transfections--
cDNA of stress-activated
protein kinase C/stress-activated protein kinase
/p54-
(GenBankTM accession number L27128) from rat brain was
inserted into the expression vector pEGFP-C3 as a
XhoI/SalI fragment. Transfection of the JNK3-EGFP
construct or the pEGFP vector was performed using the TransFast
transfection system according to the manufacturer's instructions.
Individual stable clones that expressed the JNK3-EGFP construct or the
pEGFP vector were selected and used for experiments. The same results
were obtained with three independent PC12 clones.
Neurite Outgrowth--
PC12 cells were plated and kept in
serum-containing medium for 24 h. Before the addition of NGF, the
cells were cultured in medium supplemented with 0.5% fetal calf serum
and 1% penicillin/streptomycin for 72 h. All of the experiments
with NGF were conducted with medium containing 0.5% fetal calf serum.
The percentage of cells with neurites longer than 1.5 diameters of the
cell body were counted on days 3 and 5 of the differentiation process.
The length of neurites was measured using LeicaQwin software (Leica).
Whole Cell Extracts and Nuclear Extracts--
Subconfluent PC12
cells were stimulated at different time points. Before harvesting, the
cells were washed with phosphate-buffered saline. For whole cell
extracts, the cells were resuspended in lysis buffer (20 mM
Tris, pH 7.4, 2% SDS, 1% phosphatase inhibitor, protease inhibitor),
incubated at 95 °C for 5 min, briefly sonicated, and centrifuged to
remove insoluble material (15,000 × g for 15 min). For
nuclear extracts, the cells were washed in phosphate-buffered saline
and lysed in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 1%
phosphatase inhibitor and protease inhibitor on ice for 10 min. After
centrifugation (12,000 × g for 5 min), pelleted nuclei
were washed in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 1%
phosphatase inhibitor, and protease inhibitor and resuspended in lysis
buffer (see above) and treated as described for whole cell lysates. The
protein extracts were stored at
80 °C.
Western Blots--
20 µg of total cellular proteins and 10 µg of nuclear proteins were separated on 12% SDS-polyacrylamide gels
and transferred to polyvinylidene difluoride transfer membranes. The
membranes were blocked with 4% nonfat dry milk and incubated with the
primary antibodies according to the manufacturer's recommendations.
After three washing steps with Tris-buffered saline with Tween 20, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody for 30 min. All of the Western blots were developed using the ECL chemiluminescence system and Hyperfilm ECL. Between the
stainings with phosphospecific antibodies and total kinase or total
transcription factor antibodies, the blots were stripped in 2% SDS,
62.5 mM Tris, and 100 mM 2-mercaptoethanol for
30 min at 50 °C, washed with Tris-buffered saline with Tween 20, and blocked again. All of the measurements of dual phosphorylated kinase
levels were normalized by hybridization with antibodies against total
kinase protein. To normalize for the protein content of each lane and
to confirm equal loading, all of the membranes were finally stained
with Ponceau S, scanned, and analyzed with the densitometrical software
Scion Image. All of the Western blots were exposed to film for varying
lengths of time, and only films generating subsaturating levels of
intensity were selected for densitometrical and statistical evaluation.
The bands were quantified using Scion Image software.
Statistical Analysis and Replication Rate--
Normal
distribution of the data was verified by calculating Lilliefors
probabilities based on the Kolmogorov-Smirnov test. Statistical
significance was determined using the t test for independent samples, and the respective results are displayed as the means ± S.D. All of the experiments and measurements were replicated at least
three times.
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RESULTS |
Characterization of Transfected Cells--
After the selection of
stable clones, vector- and JNK3-transfected PC12 cells were monitored
with regard to cell morphology, growth characteristics, and the
expression status of different mitogen-activated protein kinases and
their substrates. No morphological changes were detected; under normal
growth conditions, both untransfected and transfected PC12 cells had a
round or polygonal shape and did not extend processes (Fig.
1A). Apart from their similar
appearance, all of the cells displayed an almost identical growth rate
with a doubling time of ~72 h. When screened for the expression of different mitogen-activated protein kinases and their substrates, the
transfection with pEGFP or JNK3-EGFP did not cause any changes in the
amount of JNK1, JNK2, p38
, ERK1/2, or ATF-2 (Fig. 1B); neither did transfection lead to the induction of c-Jun (data not
shown). Furthermore, the amount of JNK3 protein produced by the
transfected cells was comparable with that of JNK1 and JNK2 (Fig.
1B). All of the transfected cells used for the experiments were constantly checked for well detectable green fluorescence.

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Fig. 1.
Characterization of JNK3-transfected PC12
cells. A, cell morphology of wild type (wt)
and JNK3-transfected PC12 cells. Both were kept under the same culture
conditions and displayed an indistinguishably similar phenotype.
B, Western blots of total JNK, JNK1, JNK2, ERK1/2, p38 ,
and ATF-2 in wild type (lane wt), vector-transfected
(lane ve), and JNK3-transfected (lane JNK3) PC12
cells showed no difference in protein expression. Bars, 50 µM.
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Activation of JNKs by Cellular Stress--
To investigate the
effect of different stress stimuli on the phosphorylation of all JNK
isoforms, PC12 cells were stimulated with UV irradiation (0.5, 2, or 6 J/cm2; 312 nm), H2O2 (60 µM), or paclitaxel/taxol (1 µM, 5 µM, or 10 µM) and incubated for 90 min
before protein extraction. JNK activities were analyzed by Western
blotting with antibodies directed against dual phosphorylated JNK
isoforms. UV irradiation induced a dose-dependent phosphorylation of total JNK (2-, 4-, and 6-fold for 0.5, 2, and 6 J/cm2 UV, respectively; Fig.
2A). Following transfection,
UV caused a similar activation of JNK3 (2-, 3-, and 5-fold,
respectively; Fig. 2A). In contrast,
H2O2 stimulation hardly affected JNK
phosphorylation (Fig. 2A). Taxol, which suppresses mitotic
spindle assembly and thereby induces microtubule damage, evoked a
dose-dependent response with a 2.5-3-fold activation of
all JNK isoforms (Fig. 2B). In all experiments, protein
expression did not change (Fig. 2).

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Fig. 2.
Activation of wild type JNK1/2 and
transfected JNK3 in response to UV irradiation, hydrogen peroxide, and
taxol. A, both wild type (wt) JNK1/2 and
EGFP-tagged JNK3 (83 kDa) were dose-dependently activated
(i.e. dual phosphorylated; p-JNK1/2 and
p-JNK3-EGFP, respectively) by UV irradiation (312 nm) as
compared with untreated control cells (lane C). Stimulation
with hydrogen peroxide (60 µM) caused only a weak
activation of JNKs. B, taxol (1, 5 or 10 µM)
evoked a dose-dependent response, but differences in JNK
activation between high and low doses of taxol were less pronounced
than with UV (A). With all stress stimuli, the p46 and p54
splice variants of JNK1/2 were equally activated. No changes in protein
expression of JNK1/2 or JNK3-EGFP were observed. The data are
representative of four independent experiments.
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To investigate whether the expression and activation of JNK3 affected
other stress kinases, we analyzed the phosphorylation of p38
by
Western blotting. Wild type, vector-transfected, or JNK3-transfected
PC12 cells showed no differences with regard to p38
activation (data
not shown).
Transfected JNK3 Increases the Sensitivity of PC12 Cells toward
Cell Death--
To examine whether the phosphorylation of JNK3 had any
influence on cell survival, PC12 cells were stimulated with UV,
H2O2, and taxol as described above. After
18 h (UV irradiation and H2O2) or 24 h (taxol), the cell survival rates were determined by trypan blue
staining. JNK3-transfected PC12 cells showed a significantly reduced
viability after 2 and 6 J/cm2 UV (42 and 5%) as compared
with wild type (60 and 12%) and vector-transfected cells (66 and 11%)
(p < 0.02; Fig. 3).
Thus, transfected JNK3 decreased cell survival by 30% (2 J/cm2) or 58% (6 J/cm2) compared with wild
type cells. Similarly, JNK3 significantly enhanced cell death for all
concentrations of taxol by a further 18% (1 µM), 20% (5 µM), and 33% (10 µM) as compared with wild type cells (p < 0.02; Fig. 3). In contrast, there was
no increase in cell death of JNK3-transfected cells after treatment
with H2O2 (Fig. 3). This finding shows that the
expression of JNK3 and the subsequently increased supply of JNKs did
not automatically integrate JNKs in the realization of cell death to
all stimuli but rather emphasizes the selectivity and specificity of
JNK functions. To control the cell death rates observed with trypan
blue staining, lactate dehydrogenase release assays were conducted that
confirmed the results obtained so far (data not shown).

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Fig. 3.
Survival of wild type, vector-transfected,
and JNK3-transfected PC12 cells following stimulation with UV, hydrogen
peroxide, and taxol. In response to all stress stimuli and doses
used, the survival of PC12 cells was significantly reduced as compared
with unstimulated controls (not indicated). With higher doses of UV
irradiation (2.0 and 6.0 J/cm2), a significantly larger
proportion of JNK3-transfected cells died compared with wild type and
vector-transfected cells (p < 0.02; indicated by
two asterisks). A significant increase (p < 0.02; two asterisks) in stress-induced cell death of
JNK3-transfected cells was also observed with all doses of taxol (1, 5, and 10 µM). The data represent four independent
experiments.
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JNK3 Enhances the Phosphorylation of ATF-2--
The two
transcription factors c-Jun and ATF-2 are commonly associated with the
JNK stress response. Therefore, we studied the effects of JNK3
activation on the phosphorylation of c-Jun and ATF-2 in PC12 cells
following UV irradiation (0.5, 2, and 6 J/cm2; 312 nm),
H2O2 (60 µM), and taxol (5 µM). The nuclear proteins were extracted after an
incubation time of 90 min.
ATF-2 phosphorylation was significantly increased in response to the
high UV doses and to taxol treatment in JNK3-transfected cells as
compared with wild type and vector-transfected cells (p < 0.05; Fig. 4, A and
B), whereas the expression of ATF-2 protein did not change
(Fig. 4A). Similar to H2O2-induced
cell death (see above), JNK3 transfection did not alter ATF-2
phosphorylation in response to H2O2 (Fig. 4,
A and B).

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Fig. 4.
Effect of transfected JNK3 on the
phosphorylation of ATF-2 and c-Jun in response to UV, hydrogen
peroxide, and taxol. A, ATF-2 was significantly
stronger phosphorylated in JNK3-transfected PC12 cells after higher
doses of UV (2.0 and 6.0 J/cm2) and 5 µM
taxol (*, p < 0.05 versus wild type PC12;
#, p < 0.05, and ##, p < 0.01 versus vector-transfected cells). B,
representative for differential phosphorylation of ATF-2
(p-ATF-2) in wild type (wt) and JNK3-transfected
cells (TX, taxol). No changes in the protein expression of
ATF-2 were observed. C, an increase in phosphorylated c-Jun
(p-c-Jun) in JNK3-transfected cells as compared with wild
type PC12 cells was only observed after taxol (TX)
stimulation. This increase in phosphorylated c-Jun was, however, partly
due to an increased induction of c-Jun protein expression in the
JNK3-transfected cells. The data shown represent five separate
experiments.
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Following UV irradiation and taxol stimulation, we detected a similar
increase in c-Jun protein expression and N-terminal phosphorylation in
the transfected and untransfected PC12 cells (Fig. 4C).
Normalization of phospho-c-Jun and c-Jun levels showed that the overall
increase in phosphorylated c-Jun was partly due to the enhanced
expression of the c-Jun protein. Similar to the minor effect on JNK
phosphorylation, H2O2 did not enhance c-Jun phosphorylation or protein expression, whereas taxol evoked the strongest c-Jun induction and phosphorylation in JNK3-transfected cells
(Fig. 4C).
Activation of JNK3 and JNK Targets in Response to NGF--
JNKs
have been associated with neurite outgrowth in vitro (25)
and regeneration in vivo (21, 29). To examine whether JNK3
takes part in the process of neuronal differentiation in PC12 cells, we
treated serum-starved PC12 cells with different doses of NGF (10 and 50 ng/ml) for 3 and 5 days. At these time points, JNK activities were
analyzed in whole cell extracts. JNK1 and JNK2 were phosphorylated to a
similar extent in JNK3-transfected cells (1.3- and 2.3-fold) and in
wild type cells (1.5- and 2-fold), but JNK3 showed an enhanced response
(2.5- and 3-fold) to NGF treatment (Fig.
5). JNK protein levels did not change
(Fig. 5).

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Fig. 5.
JNK3-transfected PC12 cells displayed a
stronger induction and phosphorylation of JNK targets following NGF
treatment (10 ng/ml). Both wild type JNK1/2 (wt) and
transfected JNK3 were activated (p-JNK1/2 and
p-JNK3-EGFP, respectively) after 3 and 5 days in medium
supplemented with NGF. The activation of JNK3 in response to NGF was
higher than the combined activity levels of JNK1 and JNK2. Induction of
c-Jun protein expression and phosphorylation of c-Jun
(p-c-Jun) were significantly stronger in JNK3-transfected
PC12 cells, whereas ATF-2 phosphorylation levels were similar in wild
type and transfected cells. The induction/phosphorylation of NF-H was
also significantly higher in JNK3-transfected cells.
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Because transfection of PC12 cells with JNK3 led to an altered
phosphorylation pattern of ATF-2 in response to apoptotic stress stimuli (see above), we investigated the expression and phosphorylation levels of c-Jun and ATF-2 in nuclear proteins on days 3 and 5 of the
differentiation process. Importantly, transfection with JNK3 increased
the induction and phosphorylation of c-Jun (Fig. 5), whereas ATF-2
phosphorylation was equal in untransfected and transfected cells (Fig.
5).
To further characterize the differentiation processes induced by NGF
and the involvement of JNK3, we determined the expression and
phosphorylation of the heavy unit of the neurofilament protein (NF-H),
which is a JNK substrate as well as a feature of neuronal differentiation in PC12 cells. For this purpose, we used an antibody that detected both the phosphorylated and the unphosphorylated form.
Transfection of JNK3 substantially increased the expression and/or
phosphorylation of NF-H as compared with wild type or
vector-transfected cells (Fig. 5).
JNK3 Promotes Formation and Elongation of Neurites in PC12 Cells in
Response to NGF--
In JNK3-transfected cells, treatment with 10 ng/ml NGF significantly accelerated the morphological changes linked to
the neuronal phenotype as compared with wild type and
vector-transfected cells (Fig.
6A). Thereby, transfected JNK3
was present both in the nucleus and the cytoplasm (Fig. 6B);
even though the cytoplasm contained substantially more JNK3 (Fig.
6C), the presence of JNK3 in the nucleus was repeatedly
established by microscopic analysis. This confirms the finding that
JNK3 transfection affected both cytoplasmic and nuclear JNK substrates
(Fig. 5). Analysis of the differentiation status after 3 and 5 days of
NGF application revealed an increase in both the number of neurites
(p < 0.000001; Fig.
7A) and the length of neurites
(Fig. 7B) as compared with wild type and vector-transfected cells. The enhanced length of the neurites became apparent on day 5 with a higher percentage of neurites longer than 40 µm in JNK3
transfected cells (57%) compared with wild type (32%) or vector-transfected cells (35%). A similar effect was observed with 50 ng/ml NGF (data not shown).

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Fig. 6.
Morphological changes during NGF-induced
differentiation of PC12 cells. A, in both wild type and
transfected JNK3 cells, morphological changes were induced by 10 ng/ml
NGF after 3 and 5 days, with JNK3-transfected cells showing a more
rapid appearance of neuron-like features (phase contrast
microscopy). B and C, transfected JNK3 is
present mainly in the cytoplasm but also in the nucleus (B)
and in neurites (C) (fluorescence microscopy).
Bars, 50 µM.
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Fig. 7.
Transfection of PC12 cells with JNK3
increases formation and elongation of neurites in response to NGF (10 ng/ml). A, JNK3 transfection caused a highly
significant increase in sprouting cells (p < 0.000001;
two asterisks) after 3 and 5 days of NGF treatment as
compared with both wild type (wt) and vector-transfected
samples (ve). B, distribution of neurite lengths
(µm) after 3 and 5 days of NGF application. Transfection with JNK3
caused a pronounced increase in neurite length after 5 days as compared
with wild type (wt) or vector-transfected cells
(ve).
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DISCUSSION |
The present study demonstrates that individual JNK isoforms,
e.g. JNK3-p54, can exert substantial but contrasting effects in neurons. Transfection of JNK3 into PC12 cells, which normally only
express the JNK1 and JNK2 isoforms, significantly enhanced cell death
in response to UV irradiation and taxol. In the context of NGF-induced
differentiation of PC12 cells, however, transfected JNK3 evoked a
substantial increase in the number and length of neurites compared with
untransfected cells. This functional dichotomy of JNK3 was mirrored by
a differential activation and induction of nuclear JNK substrates,
i.e. enhanced phosphorylation of ATF-2 following death
signaling in response to UV or taxol and increased expression and
phosphorylation of c-Jun during NGF-induced differentiation. The
absence of significant JNK activation or target phosphorylation in
response to the stress stimulus H2O2 supports
our hypothesis (21, 32) that JNKs, including JNK3, are not merely
injury- or stress-specific kinases but also have relevant physiological functions depending on the cellular context and the switch of nuclear
effector substrates.
JNK3 as a Mediator in Neuronal Cell Death
UV Irradiation--
We have extended the knowledge about
intracellular alterations in mammalian cells triggered by UV
irradiation (33) by novel insights into the function of JNKs. UV
irradiation activated all of the JNK isoforms and induced expression
and N-terminal phosphorylation of c-Jun. In this context, transfected
JNK3 not only increased cell death but enhanced the phosphorylation of
ATF-2, whereas c-Jun expression and activation was not affected by the
increased pool of JNK molecules.
UV irradiation is a particularly strong stimulus for both the
activation of JNKs, including JNK3, and the expression/phosphorylation of c-Jun (30, 34, 35). The fact that transfected JNK3 had no
impact on c-Jun expression and phosphorylation suggests that c-Jun
activation following UV is mediated via the Ras/ERK pathway (36, 37)
rather than by the characteristic phospho-c-Jun autoregulatory loop
(38, 39). It remains to be elucidated whether the function of the
Ras/ERK pathway is confined to the induction of c-Jun or includes a
JNK-independent N-terminal phosphorylation of c-Jun (40).
It is the ATF-2 transcription factor that serves as the predominant
nuclear receptor for JNKs in response to UV irradiation (41, 42). By
its high transactivating potential, ATF-2 can enhance or even replace
the action of c-Jun (43-46). Thus, despite the general view that JNKs
exert their degenerative-apoptotic effects via N-terminal
phosphorylation of c-Jun, the previous reports and the present
results demonstrate that the increased phosphorylation of ATF-2 is
linked to the enhanced potential of JNK3 to trigger UV-evoked cell
death, which sheds new light on stimulus-specific
neurodegenerative actions of ATF-2 downstream of JNKs. In this context,
ATF-2 might cooperate with c-Jun to induce death effectors such as Fas
ligand (15, 45, 46) and should not be overlooked as an important JNK
target in neuronal cell death. We could exclude the contribution of
p38
to the enhanced ATF-2 phosphorylation in JNK3-transfected cells,
because p38
was activated to a similar extent in wild type and
transfected PC12 cells in response to UV.
Taxol--
The chemotherapeutic agent taxol suppresses mitotic
spindle assembly and chromosome movement, which leads to cell cycle
arrest at the G2/M phase (47) and dysfunction of the
cytoskeleton. Similar to UV irradiation, treatment of PC12 cells with
taxol activated all JNK isoforms and c-Jun. Transfection with JNK3
selectively increased the phosphorylation of ATF-2, but not of c-Jun,
and in consequence significantly enhanced cell death. As reported recently, taxol is a particularly potent activator of JNKs and their
nuclear substrates c-Jun and ATF-2 (47-50). Most authors agree that
JNKs propagate cell death in response to microtubule inhibiting agents
(51-53), whereas single observations indicate a protective role for
JNKs in this context (54). In cortical neurons, taxol has been shown to
promote cell death by activating the nuclear JNK pool and c-Jun;
unfortunately, the phosphorylation of ATF-2 was not investigated
(51).
H2O2--
In our study,
H2O2 (60 µM) had only moderate
effects on both cell death and the activation of JNKs and their
targets. So far, the JNK activation by H2O2 had
not specifically been addressed in PC12 cells, but JNK activation has
been described following reactive oxygen species and redox reactions
(55). Transfected JNK3 did not alter the death rate or the
phosphorylation levels of c-Jun and ATF-2. These observations
convincingly demonstrate that the increase in JNK molecules by JNK3
transfection per se is not sufficient to influence cell
survival or activation of nuclear substrates and that the action of
JNKs in PC12 cells is highly stimulus-specific.
JNK3 Promotes Neuronal Differentiation
In response to NGF, PC12 cells differentiate into neuron-like
cells with formation and elongation of neurites. We could demonstrate that this differentiation went along with the activation of all JNK
isoforms as well as an increased expression and phosphorylation of
c-Jun. All of these effects, including the formation of neurites, were
enhanced by transfection with JNK3.
The activation of JNKs after NGF treatment in PC12 cells has already
been described (23, 25, 56), but so far, its meaning is completely
unknown. Our finding of the differentiation-triggering role of the JNK3
isoform, however, strikingly counteracts the widely accepted role of
JNK3 as a pro-degenerative effector molecule (10, 18). It deserves
major interest that the promotion of differentiation by JNK3 involves
the induction and phosphorylation of c-Jun. In both PC12 cells and
oligodendrocytes, NGF treatment results in JNK3 activation with p75 and
Rac as upstream intermediates (18, 25, 28, 35). In striking contrast to
the differentiation of PC12 cells, oligodendrocytes undergo apoptosis
in response to NGF, suggesting that the change in JNK3 signaling is
mediated either by elements downstream of Rac or by modulation of the
p75-tyrosine receptor kinase A interaction.
The enhanced induction/phosphorylation of NF-H in JNK3-transfected
cells further supports the neurite outgrowth-promoting role of JNK3.
NF-H expression and phosphorylation underlies the elongation as well as
the stabilization of neurites and facilitates its own transport.
Therefore, by inducing increased expression/phosphorylation of NF-H,
JNK3 can further advance sprouting.
The substrate switch between ATF-2 and c-Jun not only represents a
novel facet of the functional JNK repertoire but can also be considered
to be the first direct evidence of the bipartite action of JNKs. JNK
isoforms can be recruited by several scaffold proteins such as
JNK-interacting protein (59),
-arrestin (60), or I
B kinase
complex-associated protein (62), and scaffold alterations in the JNK3
signalosome might account for the substrate switch, but these
mechanisms remain to be elucidated.
The activation of c-Jun by JNK3 potentiates the
differentiating-regenerating potential of phosphorylated c-Jun in
vitro, which also holds a particular position in the cell body
response in the brain following nerve fiber damage (21, 63, 64).
Our findings support the importance of JNKs and phosphorylated c-Jun for neuronal differentiation (23, 25, 65, 66). In detail, we have
directly proven the contribution of the JNK3 isoform to both the
formation and elongation of neurites. Therefore, as discussed before
(4), the predominantly neuronal expression of JNK3 does not support the
widely expressed notion of a specific neurodegenerative role as it was
described in neurons following, for example, UV irradiation in
vitro (16), kainate excitotoxicity in vivo (10, 16), or
withdrawal of trophic support (19) or at least partially following DNA
damage (17).
In the present study, we have shown that a single splice form of JNK3
(JNK-p54) has the potential to exert pivotal effects in a defined cell
culture system such as PC12 cells. On the one hand, it enhances cell
death caused by UV irradiation and taxol treatment, and on the other
hand, transfected JNK3 increases the number and length of neurites in
PC12 cells. The underlying substrate switch of JNK3 sheds new light on
the role of ATF-2 in neurodegeneration. The functional versatility of
JNK3 in physiological and pathological situations requires a
substantial revision of the general notion of JNK3 as a neuronal stress kinase.