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A single c-Jun N-terminal kinase isoform (JNK3-p54) is an effector in both neuronal differentiation and cell death.

The c-Jun N-terminal kinases (JNKs) mediate degeneration and apoptosis in the brain. Particularly, JNK3 is considered to be a degenerative enzyme with c-Jun as a relevant substrate. The contribution of individual JNK isoforms, however, to pathological as well as to physiological processes remains to be defined. To analyze the effects of a single JNK isoform on neuronal cell death and differentiation, we transfected PC12 cells, which normally express only JNK1 and JNK2, with JNK3-p54. Transfected JNK3 significantly enhanced cell death after UV irradiation (0.5-6 J/cm(2)) and paclitaxel/taxol treatment (1-10 microm). In contrast, in the context of nerve growth factor-induced (10 or 50 ng/ml) differentiation of PC12 cells, JNK3 expression significantly increased the number and length of neurites. This functional dichotomy of JNK3 was mirrored by differential activation and induction of nuclear JNK substrates; although activating transcription factor-2 phosphorylation was enhanced by death signaling in response to UV and taxol, c-Jun protein expression and N-terminal phosphorylation were increased during nerve growth factor-induced differentiation. The absence of significant JNK activation or target phosphorylation in response to H(2)O(2) (60 microm) further supports the hypothesis that JNK isoforms are not merely injury- or stress-specific kinases but also have context-specific physiological functions.

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)(14)(15)(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 p75mediated 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)(23)(24)(25)(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 H 2 O 2 . 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).
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% CO 2 . Transfected cell clones were kept in the presence of 500 g/ml G418 to maintain selection.
Plasmids and Transfections-cDNA of stress-activated protein kinase C/stress-activated protein kinase ␤/p54-␤ (GenBank TM 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 ϫ 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 MgCl 2 , 1% phosphatase inhibitor and protease inhibitor on ice for 10 min. After centrifugation (12,000 ϫ g for 5 min), pelleted nuclei were washed in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , 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 Ϫ80°C.
Western Blots-20 g of total cellular proteins and 10 g 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 Ϯ S.D. All of the experiments and measurements were replicated at least three times.

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 ϳ72 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.
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, H 2 O 2 , and taxol as described above. After 18 h (UV irradiation and H 2 O 2 ) 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/cm 2 UV (42 and 5%) as compared with wild type (60 and 12%) and vector-transfected cells (66 and 11%) (p Ͻ 0.02; Fig. 3). Thus, transfected JNK3 decreased cell survival by 30% (2 J/cm 2 ) or 58% (6 J/cm 2 ) compared with wild type cells. Similarly, JNK3 significantly enhanced cell death for all concentrations of taxol by a further 18% (1 M), 20% (5 M), and 33% (10 M) as compared with wild type cells (p Ͻ 0.02; Fig. 3). In contrast, there was no increase in cell death of JNK3-transfected cells after treatment with H 2 O 2 (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).
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/cm 2 ; 312 nm), H 2 O 2 (60 M), and taxol (5 M). 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 JNK3transfected cells as compared with wild type and vector-transfected cells (p Ͻ 0.05; Fig. 4, A and B), whereas the expression of ATF-2 protein did not change (Fig. 4A). Similar to H 2 O 2 - induced cell death (see above), JNK3 transfection did not alter ATF-2 phosphorylation in response to H 2 O 2 (Fig. 4, A and B).
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, H 2 O 2 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).
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 Ͻ 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 m 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).

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 H 2 O 2 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 irradi-ation (41,42). By its high transactivating potential, ATF-2 can enhance or even replace the action of c-Jun (43)(44)(45)(46). Thus, despite the general view that JNKs exert their degenerativeapoptotic 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 G 2 /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)(48)(49)(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).
H 2 O 2 -In our study, H 2 O 2 (60 M) had only moderate effects on both cell death and the activation of JNKs and their targets. So far, the JNK activation by H 2 O 2 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 JNK3transfected 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 IB 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.